Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae

We compared the humoral immune response of mice protected against Schistosoma mansoni by vaccination with radiation-attenuated cercariae to that of patently infected mice, and we identified antigens that elicit a greater, or unique, immune response in the vaccinated mice. These comparisons were based upon radioimmunoprecipitations and immunodepletion of [35S]methionine-labeled schistosomular and adult worm polypeptides, followed by one- and two-dimensional polyacrylamide gel analyses. The humoral responses of patently infected mice and of mice vaccinated once were remarkably similar and were directed against schistosome glycoproteins ranging in molecular size from greater than 300 to less than 10 kDa. Exposing mice to a second vaccination resulted in a marked change in the immune response, to one predominantly directed toward high molecular size glycoproteins. Sequential immunodepletion techniques identified five schistosomular and seven adult worm antigens that showed a greater or unique immunogenicity in vaccinated mice as compared with patently infected mice. These adult worm antigens were purified by preparative sequential immunoaffinity chromatography and used to prepare a polyclonal antiserum, anti-irradiated vaccine. This antiserum bound to the surface of live newly transformed and lung-stage schistosomula, as assessed by immunofluorescence assays, and was reactive with a number of 125I-labeled schistosomular surface polypeptides, including a doublet of 150 kDa that was also recognized by sera of vaccinated mice but not by sera of patently infected mice.     

In situ pulmonary responses of T cell and macrophage subpopulations to a challenge infection in mice vaccinated with irradiated cercariae of Schistosoma mansoni

Cellular events related to the resistance induced by radiation-attenuated cercariae of Schistosoma mansoni were determined immunocytochemically in the lung tissues of mice. Thy-1, CD4, CD8, Mac-1 MOMA-1, MOMA-2, and Ia antigens were identified on cryostat sections by the immunogold-silver staining technique with specific monoclonal antibodies. In mice vaccinated with irradiated cercariae and challenged with normal cercariae, the number of Thy-1+ and CD4+ lymphocytes was increased dramatically relative to the normal numbers both in perivascular tissues and in focal cellular aggregates in the parenchyma of the lungs. A high ratio of CD4+/CD8+ T cells was noted in the aggregates, both in perivascular tissues and in the foci. Macrophages showing positive reactions for Mac-1, MOMA-1, MOMA-2, and Ia also infiltrated the foci. In control mice that were unvaccinated and challenged, foci showing positive reactions for the lymphocyte subpopulations barely were detectable in the lungs by day 14. The numbers of Thy-1+, CD4+, and CD8+ cells and the CD4+/CD8+ ratio in controls were considerably less than those in vaccinated/challenged mice over the period of observation. In conclusion, pulmonary cellular aggregates in vaccinated and challenged mice were composed mainly of Thy-1+ and CD4+ cell populations characteristic of delayed-type hypersensitivity (DTH) reactions. Thus, Thy-1+ and CD4+ cells in the lungs of vaccinated mice may be involved in the elimination of challenge parasites through DTH reactions.     

Intraspecific cross-protection in mice immunized with irradiated Schistosoma mansoni cercariae

Several laboratory-maintained strains of Schistosoma mansoni were tested for their relative immunogenicity or susceptibility to anti-schistosome immunity in irradiated cercaria-immunized mice. A total of 11 strains and substrains were used; 7 were of Puerto Rican origin, 3 from Brazil, and 1 from Egypt. Mice were immunized by percutaneous exposure to 50-krad-irradiated cercariae. Immunity was assessed following challenge with cercariae of the homologous or a heterologous strain. The results showed that the choice of either the challenge or immunizing strains was not critical in the development of significant levels of protection. Extensive degrees of cross-protection developed in all intrastrain combinations tested.     

Pulmonary leukocytic responses are linked to the acquired immunity of mice vaccinated with irradiated cercariae of Schistosoma mansoni

Pulmonary cellular responses in C57BL/6 mice exposed to Schistosoma mansoni have been investigated by sampling cells from the respiratory airways with bronchoalveolar lavage. Mice exposed to cercariae attenuated with 20 krad gamma-radiation developed stronger and more persistent pulmonary leukocytic responses than animals exposed to equal numbers of normal parasites. Although vaccination with irradiated cercariae also stimulated T cell responses of greater magnitude and duration than normal infection, the lymphocytic infiltrate elicited by each regimen did not differ substantially in its composition, 5 wk after exposure. Studies with cercariae attenuated by different treatments established that a link exists between the recruitment of leukocytes to the lungs of vaccinated mice and resistance to reinfection. There was a strong association between pulmonary leukocytic responses and the elimination of challenge infections by vaccinated mice. Animals exposed to irradiated cercariae of S. mansoni were resistant to homologous challenge infection but were not protected against Schistosoma margrebowiei. Homologous challenge of vaccinated mice stimulated anamnestic leukocytic and T lymphocytic responses in the lungs, 2 wk postinfection, but exposure of immunized animals to the heterologous species failed to trigger an expansion in these populations of cells. Our studies indicate that pulmonary leukocytes and T lymphocytes are intimately involved in the mechanism of vaccine-induced resistance to S. mansoni. It remains unclear whether these populations of cells initiate protective inflammatory reactions against challenge parasites in the lungs, or accumulate in response to the activation of the protective mechanism by other means.     

The ability of fractionated sera from animals vaccinated with irradiated cercariae of Schistosoma mansoni to transfer immunity to mice

To study the role of IgG and IgM isotypes found in sera of mice or rabbits immunized with irradiated cercariae in schistosome immunity, the respective sera were fractionated by protein A chromatography. Both the protein A-bound and unbound fractions of vaccinated mouse serum (VMS) showed reactivities in ELISA assay using NP-40 membrane extracts of 3-hr schistosomula as antigens and in indirect immunofluorescence assay (IIF) using live 3-hr schistosomula. Both the protein A-bound and unbound fractions possessed high levels (84% and 76%, respectively) of complement-mediated cytotoxicity against schistosomula in vitro. The IgG- and IgM-containing fractions each conferred passive protection (30% and 20%) against challenge infection, although at a lower level when compared to unfractionated VMS (42%). These data demonstrate that in the mouse model both IgG and IgM can recognize epitopes on the surface of schistosomula, mediate cytotoxicity in vitro, and provide passive protection in vivo. Similarly, the protein A-bound and unbound fractions of vaccinated rabbit serum (VRS) were also shown to be positive in ELISA and IIF. The IgG- and IgM-containing fractions each possessed high levels (95% and 85%, respectively) of complement-dependent cytotoxicity against schistosomula in vitro. In contrast to VMS fractions, the IgG fraction of VRS conferred a similar level (28%) of in vivo protection as unfractionated VRS when injected into mice no later than 6 days after challenge. Moreover, the IgG fraction of VRS was still able to provide passive protection to mice when given as late as 15 days postinfection, but failed to confer protection when injected at 24 or 35 days postinfection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibody to IFN-gamma modifies pulmonary inflammatory responses and abrogates immunity to Schistosoma mansoni in mice vaccinated with attenuated cercariae.

In C57Bl/6 mice vaccinated with a single dose of attenuated cercariae of Schistosoma mansoni, the major site of immune elimination of intact challenge parasites is the lungs. The effector mechanism involves the formation of focal inflammatory responses throughout the pulmonary tissues. These foci are rich in CD4+ T cells, believed to be memory:effector cells of the Th1 type. To investigate the role of IFN-gamma in these inflammatory responses, vaccinated mice were treated with neutralizing mAb. Administration on days 4, 8, 12, and 16 post-challenge, the period over which elimination of challenge parasites takes place in the lungs, gave an average 89.5% abrogation of protective immunity. Analysis of pulmonary cell populations recovered by bronchoalveolar lavage from treated nonimmune mice at day 14 post-challenge revealed a sharp increase in pulmonary eosinophilia, relative to intact vaccinated and challenged animals. The inverse relationship between eosinophilia and protection suggests that eosinophils do not play a vital role in the immune effector mechanism in this model. Pulmonary foci of treated mice were larger, less compact, and of different cellular composition from those of control groups. They contained increased numbers of eosinophils, together with numerous multinucleated giant cells. The effects observed in the anti-IFN-gamma mAb-treated mice, together with the maintenance of MHC class II expression on alveolar macrophages in these animals, could all be explained by the production of IL-4 and other Th2 cytokines. Thus, neutralization of IFN-gamma during challenge responses may shift the Th balance towards domination by the Th2 subset.     

Schistosoma mansoni: influence of immunization and challenge schedules on the sites and mechanisms of resistance in CBA/Ca mice vaccinated with highly irradiated cercariae

These studies address current controversies over the site(s) of challenge attrition in the murine irradiated vaccine model of immunity to Schistosoma mansoni. Two possibilities have been investigated. Firstly, that the site of death of the radiation-attenuated schistosomes used to vaccinate the mice may vary in different laboratories and secondly, that the skin sites selected for presentation of the immunizing and challenge parasites may influence the final site at which immunity is effected (i.e. ear/abdomen vs tail/tail). The migration of radiolabelled cercariae exposed to 0, 20 or 50 krad of gamma irradiation from the NIMR 60Cobalt source was examined in CBA/Ca mice by squashed organ autoradiography. Unirradiated parasites all migrated from the skin to the lungs, and 65% moved on to the liver. Migration of parasites attenuated by exposure to 20 krad of gamma irradiation was delayed, but 76% finally reached the lungs; only 1% was recruited to the liver. The majority of 50 krad attenuated parasites died in the skin, with only 4% accomplishing migration to the pulmonary vasculature. The major site of death (and by implication of antigenic stimulation) of the 20 krad attenuated NIMR strain S. mansoni used routinely for vaccination purposes in our laboratory, is thus the lungs, a finding that does not explain the fact that immunity is mediated primarily in the skin in our model system. Site elimination experiments and squashed organ autoradiography showed conclusively that, irrespective of the skin sites chosen for presentation of the immunizing and challenge population of worms, NIMR challenge parasites are killed predominantly in the skin of vaccinated CBA/Ca mice. Moreover, qualitative and quantitative histological examination of the challenged tail skin of vaccinated mice revealed that inflammatory reactions comprising mononuclear cells and eosinophils develop in this site and function to trap and eliminate challenge larvae, despite a reported reduction in antigen presenting cells in this region.     

Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae

The humoral immune responses of mice patently infected with Schistosoma mansoni and of mice vaccinated with radiation-attenuated cercariae were compared by radioimmunoassays and one- and two-dimensional polyacrylamide gel analyses of radioimmunoprecipitates. The binding observed with antibodies of mice vaccinated twice with radiation-attenuated cercariae over a period of 7 to 11 wk was less than 50% of the binding observed with antibodies of mice patently infected for 20 wk, but three to four times greater than that obtained with antibodies of mice infected for 6 wk, irrespective of whether the test antigen extracts were derived from schistosomula or adult worms. Sera of vaccinated mice precipitated a restricted number of predominantly high m.w. glycoproteins of both schistosomula and adult worms metabolically labeled with [35S] methionine. Each of the glycoproteins of 36 hr in vitro-cultured schistosomula that was precipitated by the sera of vaccinated mice was also precipitated by sera of infected mice. In contrast, sera of vaccinated mice uniquely precipitated a 38,000 m.w. glycoprotein of schistosomula cultured for 5 days and a 94,000 m.w. glycoprotein of adult male worms. Although radiation-attenuated larvae do not reach the adult stage, mice vaccinated with these still elicit a strong immune response against egg glycoproteins. In particular, an egg glycoprotein of 85,000 to 70,000 and isoelectric point of 4.8 showed an enhanced reactivity with sera of vaccinated mice in comparison with infected mice. These results show that the antibody response in mice vaccinated with radiation-attenuated larvae differs qualitatively and quantitatively from that of infected mice.     

Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae. IV. Analysis of the role of IgE antibodies and mast cells

Mice resistant to challenge infection with Schistosoma mansoni by vaccination with highly irradiated cercariae were examined for the presence of circulating IgE antibodies and peritoneal mast cells sensitized against schistosome antigens. Significant levels of SWAP- or CAP-specific IgE antibodies could not be detected by solid phase radioimmunoassay in the sera of C57BL/6 mice during the first 6 wk after vaccination. Similarly, heatlabile antibodies capable of passively sensitizing normal mast cells for degranulation in response to SWAP could not be identified in the same sera. In contrast, peritoneal mast cells harvested from C57BL/6 mice 2 wk or later after vaccination gave strong degranulation responses when challenged with SWAP or CAP. Thus, vaccination with irradiated cercariae induces an unusual form of immediate-type hypersensitivity in which mast cells become sensitized in the absence of detectable circulating IgE antibodies. Mice deficient in mast cells (W/Wv mutant strain) were observed to develop the same resistance to challenge infection after vaccination with irradiated cercariae as nondeficient littermates. Similarly, vaccinated SJL/J mice were found to mount an extremely weak IgE response as measured by mast cell degranulation yet displayed the same level of resistance to challenge infection as other inbred mice developing potent mast cell responses. These findings argue that IgE antibodies and mast cells are not essential components in the effector mechanism of irradiated vaccine-induced immunity against schistosome infection.     

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae. International Journal for Parasitology16: 491–510. Naive guinea pigs and guinea pigs vaccinated 4 weeks previously with highly irradiated cercariae were challenged percutaneously with normal cercariae. Skin samples from the challenge site were then harvested at varying times to provide histological, quantitative and ultrastructural data on the respective cellular responses to cercarial invasion. The primary cutaneous reaction was characterised by neutrophils; these cells reached peak numbers (16% of total cells) by 18 h. Eosinophils and basophils made only a small contribution to the infiltrate (2.9 and 5.7% respectively). Some basophils showed evidence of anaphylactic degranulation, others seemed to be damaged, but most appeared normal. Mononuclear cells of varied morphology were present at all times, but activated fibroblasts were prominent, and collagen deposition increased with time. Degranulating mast cells were recognised at 24 and 48 h. Dead schistosomula were never seen in naive-challenged skin, although one or two of the observed larvae showed minor tegumental abnormalities. In vaccinated guinea pigs, the cutaneous cellular response to challenge was significantly enhanced, with basophils dominating the reaction (33% of total cells at 24 h). Many basophils were already degranulating by the anaphylactic mechanism at 3 h post challenge, and free basophil granules were seen frequently. Both intact cells and free granules congregated in close proximity to invading larvae. Eosinophils were also present in greater numbers at secondary reaction sites, but they never exceeded 6% of the total infiltrate. Mononuclear cells believed to be immature eosinophils were prominent from 3 h. For the first time, the mechanism of eosinophil attachment and degranulation onto a multicellular target, described previously only from in vitro investigations, was recognized in vivo. Neutrophil numbers matched those recorded in naive-challenged skin at 3 and 6 h, but declined thereafter, while mast cells were seen degranulating at these early times. Mononuclear cells again presented a variety of morphological appearances; of particular note were large cells that had phagocytosed debris and were presumed to be macrophages, and small rounded cells with scant cytoplasm and few organelles, that may have been lymphocytes. By 12 h, large areas of the dermis had become severely disorganised and numerous, free basophil granules were distributed amongst the other cellular constituents. Dead schistosomula, denuded of tegument, were clearly recognised at 6 h, and such individuals invariably had neutrophils attached to their exposed muscle layers. Since dead schistosomula were not identified in naive-challenged guinea pig skin, it is concluded that a percentage of the challenge larvae, however small, is preferentially killed in the skin of the vaccinated animals.     

Schistosoma mansoni: vaccination of mice with cryopreserved irradiated schistosomules.

In our earlier experiments, NIH/Nmri (CV) mice developed protective immunity to a Schistosoma mansoni cercarial challenge when previously exposed percutaneously to highly ⁶⁰Co-irradiated homologous cercariae. Experiments reported here were conducted to assess the immunogenicity of unfrozen and frozen and thawed schistosomules derived from ⁶⁰Co-irradiated cercariae (irradiated schistosomules). Immunization of NIH/Nmri (CV) mice by ⁶⁰Co-irradiated unfrozen schistosomules reduced worm burdens from a subsequent percutaneous challenge with normal cercariae by 41 to 72%. Immunogenicity was not narrowly dependent on irradiation dose rates between 1 and 8 kR/min, or on the total dose of irradiation given the schistosomules between 25 and 50 kR. Comparable protective immunity developed after injection of irradiated schistosomules which had been frozen to ?196 C in liquid nitrogen and thawed. Cryopreservation appears to offer a solution to the problem of storage of attenuated, immunogenic S. mansoni schistosomules.     

Comparison of Th1- and Th2-associated immune reactivities stimulated by single versus multiple vaccination of mice with irradiated Schistosoma mansoni cercariae.

Mice immunized against Schistosoma mansoni by a single percutaneous exposure to radiation-attenuated parasite larvae demonstrate partial resistance to challenge infection that has been shown to correlate with development of cell-mediated immunity, whereas mice hyperimmunized by multiple exposure to attenuated larvae produce antibodies capable of transferring partial protection to naive recipients. Measurement of Ag-specific lymphokine responses in these animals suggested that the difference in resistance mechanisms may be due to the differential induction of Th subset response by the two immunization protocols. Thus, upon Ag stimulation, singly immunized mice predominantly demonstrated responses associated with Th1 reactivity, including IL-2 and IFN-gamma production, whereas multiply immunized animals showed increased IL-5, IL-4, and IgG1 antibody production associated with enhanced Th2 response. These responses demonstrated some degree of organ compartmentalization, with splenocytes demonstrating higher Th1-related lymphokine production and cells from draining lymph nodes showing stronger proliferation and Th2 type reactivity. However, hyperimmunized mice also continued to demonstrate substantial Th1-associated immune reactivity. Moreover, in vivo Ag challenge elicited activated larvacidal macrophages in hyperimmunized animals. These observations indicate that protective cell-mediated mechanisms associated with induction of CD4+ Th1 cell reactivity predominate in singly vaccinated mice. Further vaccination stimulates Th2 responses, such as enhanced IgG1 production, that may also contribute to protective immunity.     

Schistosoma mansoni: parasitology and immunology of baboons vaccinated with irradiated cryopreserved schistosomula

Young, captivity-born male baboons (Papio cynocephalus) were vaccinated with γ-irradiated (500 Gy) cryopreserved Puerto Rican strain schistosomula of S. mansoni. Protection against heterologous, normal Kenyan Strain S. mansoni challenge infection was erratic and partial; and two putative correlates of immunity, reduced worm fecundity and change in worm location (anterior shift) were not observed. However, immunization of baboons with this vaccine resulted in a stimulated immune system. Both cellular and humoral anamnesis were demonstrable in vaccinated-challenged baboons. Schistosome infection-associated IgM hypergammaglobulinema was also greatly reduced in vaccinated-challenged baboons. On the other hand, IgG antibodies to adult, egg, and cercarial antigens were increased after challenge infection in preimmunized baboons. Vaccination appears to have resulted in a redirection of the immune system into anti-parasite channels, but this more specific immune response was insufficient to confer good protection against challenge infection in this experiment. The dampening effect of the vaccine on the hypergammaglobulinemia of schistosomiasis is another candidate for a possible “anti-pathogenesis” effect of irradiated schistosome larval vaccines, to be added to the reduced granuloma size already reported (Damian, Roberts, Powell, Clark, Lewis &; Stirewalt, 1984) to occur in these vaccinated baboons. Together, they could increase the potential benefit of live, irradiated vaccines for schistosomiasis. Human trials with vaccines only partially protective against challenge infections may therefore ultimately be warranted.     

Mechanisms of protective immunity against Schistosoma mansoni infection in mice vaccinated with irradiated cercariae: I. Analysis of antibody and T-lymphocyte responses in mouse strains developing differing levels of immunity

The kinetics of cellular and humoral responses directed against schistosomula were examined in mice of three inbred strains which demonstrate differences in the degree of resistance induced by immunization with irradiated cercariae. T-Cell reactivity was observed during the first 4 weeks after vaccination but declined to control levels thereafter. Anti-schistosomulum antibody was first detected 2 weeks after vaccination, peaked by 6 weeks, and persisted as late as 15 weeks. In sera obtained at 6 weeks, antibody activity was detected in affinity chromatography-purified fractions containing IgM, IgA, IgG₁, IgG_2a, and IgG₃ immunoglobulins. In general, the cellular and humoral responses observed in C57B1/6J mice, which consistently developed a high level of immunity after vaccination, were not significantly different from those observed in C3H/HeJ or CBA/J mice, which achieved only low to moderate levels of immunity. Thus, although antibody production appears to correlate more closely than T lymphocyte responsiveness with the typical long-term resistance pattern observed in this model, the absence of striking differences in parasite-specific antibody levels between mice of these different strains suggests that additional mechanisms may be involved in the development of immunity after vaccination.     

Detection of Schistosoma mansoni circulating cathodic antigen for evaluation of resistance induced by irradiated cercariae.

The appearance of serum levels of circulating cathodic antigen (CCA) detectable by a monoclonal antibody (mAb) (5H11) antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) system was evaluated during acute Schistosoma mansoni infections in female CF1 mice exposed to either 100 or 25 cercariae. Measurable CCA levels occurred in these groups at 5 and 7 wk after infection, respectively. The kinetics of appearance of CCA were thus related to the intensity of infection. The level of resistance developed by female C57BL/6 mice upon immunization with irradiated cercariae, as expressed by both worm burden and CCA levels after cercarial challenge was evaluated. Immunization conferred 44% protection against the challenge infection, and the level of CCA detected in the sera of the control group was significantly (P less than 0.02) higher than that found in the sera of the immunized group, 6 wk after challenge. These results demonstrate that CCA detection by the 5H11 mAb antigen-capture sandwich ELISA can reflect vaccine-induced resistance against S. mansoni. Localization studies showed that 5H11 reacts with a CCA epitope in the adult worm gut and to a lesser extent with the male tegument. Adaptations of this and other antigen detection systems may prove useful in monitoring the efficacy of developmental vaccines, an ability that may be essential for the extension of such studies to humans.     

Vaccination with irradiated cercariae of Schistosoma mansoni preferentially induced the accumulation of interferon-gamma producing T cells in the skin and skin draining lymph nodes of mice

Cytokine response to schistosomula of Schistosoma mansoni was evaluated in the skin of mice during the initial 72 h following infection. These studies showed a significant increase in the levels of IL-4 and IL-10 message in the skin in areas of cercarial penetration. The IL-4 message was detectable in the skin as early as 8 h after infection and the message for IL-10 appeared from 16 h after infection. However, mRNA for IFN-gamma was undetectable in the skin samples for up to 72 h after infection with normal cercariae. In sharp contrast, vaccination with irradiated cercariae induced IFN-gamma and IL-2 responses in the skin within 24 h. Analysis of the cytokine profile of cells isolated from the skin during these early time points showed that T cells are probably not a source of IL-4 or IL-10 in the skin of mice infected with normal cercariae. However, in vaccinated animals, the majority of the IFN-gamma is derived from skin-residing T cells. In vaccinated animals, responses in the skin were mirrored in the skin-draining lymph nodes as well. Analysis of the CD4/CD8 ratio showed a significant decrease in the skin following vaccination suggesting an increase in CD8+ cells. Interestingly however, when vaccinated animals were challenged with normal cercariae, there was a significant reduction in IFN-gamma response in the skin and its draining lymph nodes. These results show that vaccination with irradiated cercariae of S. mansoni, preferentially induce the accumulation of IFN-gamma producing T cells in the skin and skin-draining lymph nodes of mice.