Irradiated Schistosoma Research Articles

Assessment of the Diagnostic Performance of Normal and Irradiated Schistosoma mansoni Cercarial Antigens using ELISA

Assessment of the Diagnostic Performance of Normal and Irradiated Schistosoma mansoni Cercarial Antigens using ELISA

Evaluation of Gamma Radiation-Attenuated Schistosoma mansoni Cercarial Antigens for Immunodiagnosis

DEVELOPMENT of sensitive and specific diagnostic tools for early diagnosis and treatment has an important necessity. This study focuses on identifying and evaluating immunodiagnostic potential of irradiated Schistosoma mansoni cercarial antigens by immunoblot analysis using infected human sera. Forty two serum samples were divided into: 14 sera from S. mansoni infected patients (Group I), 18 sera of patients with other parasitic diseases (Group II) and 10 sera from healthy individuals (Group III). Characterization of normal and 0.4Gy gamma radiation-attenuated cercarial antigens (Cer. Ags) was conducted using Gel Electrophoresis (SDS- PAGE). Normal Cer. Ags was resolved into six individual bands of 205, ≈160, 97, 66, 43 and ≈38kDa while irradiated Cer. Ags. showed same bands except 66kDa, in addition to two more bands which were ≈50 and 24kDa. Immunoblot analysis of irradiated cercarial antigens using pooled sera of patients with schistosomiasis mansoni identified four bands two like normal one, 97kDa and ≈32kDa, in addition to ≈50kDa and 43kDa bands. Total IgG immunoblotting using irradiated cercarial antigens with individual sera of patients with schistosomiasis and other parasites revealed that higher diagnostic accuracy was achieved by both 97kDa and 24kDa with 78.6%. Both 97 and 43kDa bands gave a higher sensitivity of 64.3% while 24kDa bands achieved the highest specificity of 89.3%. It could be concluded that total immunoblotting using irradiated cercarial antigens at 97kDa and 24kDa proved to be valuable as diagnostic tests for human schistosomiasis.

Schistosoma mansoni: antigen-presenting cells emigrating from skin exposed to attenuated cercariae activate lymphoid cells and transfer protection in C57B1/6 mice.

C57B1/6 mice develop significant levels of protection to a challenge infection after percutaneous exposure to irradiated Schistosoma mansoni cercariae. Although some circumstantial evidence has suggested that antigen-presenting cells (APCs) within the skin play a role in priming anti-schistosomula effector mechanisms, no direct evidence has been presented. In this study, we describe efforts to directly test whether skin-resident APCs exposed to irradiated cercariae are capable of mediating responses consistent with previously proposed mechanisms associated with delayed-type hypersensitivity reactions. We demonstrate that a population of APCs emigrates from the skin after percutaneous vaccination and that these cells are able to induce proliferation of S. mansoni-specific lymphocytes. We describe our experiments conducted to confirm that proliferation is dependent on major histocompatibility complex (MHC) Class-II interactions and cell-to-cell contact between APCs and lymphocytes. Immunohistological staining of emigrating cells revealed a population of large MHC Class-II+ cells with a morphology characteristic of mature dendritic cells. On recovery and adoptive transfer into naive mice, these cells demonstrated the ability to mediate protection to a challenge infection at levels similar to those in percutaneously vaccinated controls. This confirms that cutaneous APCs can initiate anti-schistosomula effector mechanisms in C57B1/6 mice after percutaneous vaccination.

Schistosoma TOR (trispanning orphan receptor), a novel, antigenic surface receptor of the blood-dwelling, Schistosoma parasite

Sh-TOR is a novel, putative three transmembrane domain receptor of Schistosoma haematobium, which has no extensive homology to any other known protein. The 0.86 kb open reading frame was found to encode a novel protein, 286 amino acids long and of 32 kDa. It was shown that Sh-TOR can be phosphorylated on tyrosine and the protein sequence reveals a long cytoplasmic tail with several consensus phosphorylation sites for enzymes which characteristically associate with membrane receptors. The proposed topology of Sh-TOR, based on antibody recognition of transfected Sh-TOR, predicts that the amino terminus is extracellular and the carboxyl terminus intracellular. Sh-TOR is a non-glycosylated protein found in the surface tegumental plasma membrane, and tegumental surface pits of adult schistosomes. The 1.35 kb transcript was most highly expressed in the larval stage, which is more susceptible to immune attack. A TOR homologue from Schistosoma mansoni is also described. A homologue from Trypanosoma cruzi, another human parasite was also isolated, but not from the free-living nematode Caenorhabditis elegans. Recombinant Sh-TOR is specifically recognised by a passively protective serum, from baboons vaccinated with irradiated Schistosoma parasite. Together with its surface location, this means that Sh-TOR is also a potential vaccine candidate molecule.

In the absence of IL-12, the induction of Th1-mediated protective immunity by the attenuated schistosome vaccine is impaired, revealing an alternative pathway with Th2-type characteristics.

Vaccination of mice with irradiated Schistosoma mansoni larvae confers high levels of immunity which is mediated by Th1-type lymphocytes. To investigate a possible role for IL-12 in the induction of protection, we have compared the immune response of IL-12 p40-deficient (KO) mice and their C57BL/6 (WT) counterparts following vaccination. Cultured lymph node cells from KO mice had markedly altered cytokine profiles with significantly decreased production of IFN-gamma increased IL-4. Correspondingly, KO mice had enhanced levels of IgE. After challenge, cells recovered from the lungs of KO mice secreted abundant IL-4 and IL-5 but little IFN-gamma, while flow cytometric and histological analysis of lung cell populations recorded a very high proportion of eosinophils. The levels of protection in KO mice were substantially lower than in their WT counterparts, demonstrating the importance of IL-12 and Th1-mediated immune responses. This conclusion is reinforced by the administration of rIL-12 to KO mice immediately after vaccination which led to increased IFN-gamma and the restoration of protective immunity. Nevertheless, the data also indicated that the limited levels of protection induced in KO mice occur via an IL-12-independent pathway, possibly mediated by Th2 cells.

Th1 cytokine mRNA expression dominates in the skin-draining lymph nodes of C57BL/6 mice following vaccination with irradiated Schistosoma mansoni cercariae, but is down-regulated upon challenge infection.

Vaccination of C57BL/6 mice with irradiated cercariae of Schistosoma mansoni results in the induction of high levels of immunity to subsequent infection. The events occurring in the lymph nodes draining the exposure site have been analysed ex vivo by reverse transcription-polymerase chain reaction (RT-PCR) and the timing of cytokine gene expression following exposure has been established. After vaccination, spatial separation of the T-helper type 1 (Th1) and Th2 responses was evident, with interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and IL-12 mRNA peaking earlier than mRNA for IL-4, IL-5 and IL-10. In contrast to the profiles observed post-vaccination, following challenge the IL-4 mRNA was predominant in the draining lymph nodes, with IFN-gamma message levels barely detectable above the naive level. These observations are confirmed by the analysis of IL-4 and IFN-gamma mRNA using competitive PCR. From these studies it is clear that irradiated cercariae are more able to promote a protective Th1 response, with normal parasites eliciting higher IL-4 and IL-5 expression upon both primary and secondary stimulation.

Sehistosoma Mansoni in the Baboon: Modulation of Pathology After Vaccination with Polyclonal Anti‐Idiotypic Antibodies

Vaccination of five baboons with an anti-idiotypic vaccine to irradiated Schistosoma mansoni cercariae resulted in nearly 19% protection compared to 39% protection conferred to five baboons vaccinated with an irradiated vaccine. Vaccination with the anti-idiotypic antibodies resulted in a significant reduction of pathology and granuloma size following challenge with live unattenuated cercariae. Results presented in this work are considered highly significant because the anti-idiotypic vaccine markedly influenced schistosomiasis morbidity which is the main consideration in this disease.

Schistosoma haematobiumin the baboon (Papio anubis): assessment of protection levels against either a single mass challenge or repeated trickle challenges after vaccination with irradiated schistosomula

Baboons vaccinated intramuscularly with three times 9000 20 krad irradiated Schistosoma haematobium schistosomula at monthly intervals were exposed percutaneously to either a single mass challenge of 3000 (VMC) or ten, weekly trickle challenges of 300 (VTC) normals S. haematobium cercariae. Unvaccinated mass (MCC) or trickle (TCC) challenge controls were exposed simultaneously. Faecal and urine egg production was delayed in the vaccinated groups which also had reduced adult, particularly female, worm recoveries. Total faecal, urine and tissue eggs were lower in the vaccinated groups, as also were the size of granulomata and the gross pathology and severity of inflammatory responses in the bladder and ureters, except for an increased proportion of tissue eggs in the livers of vaccinated animals. Differences in pathology between groups were less marked in the other organs. Most indices were reduced in the trickle versus the mass challenge control groups. Some of these trends were statistically significant, mostly in the trickle vaccination group (VTC), but others were not. Compared with the appropriate unvaccinated controls, the percentage reduction for the trickle challenge (VTC) group (73%) was three times greater than that of the mass challenge (VMC) group (23%). Overall, the protective effect of vaccination was more clearly demonstrated in the trickle than in the mass challenge groups. This conclusion is based on a single experiment. Nevertheless, because trickle infections probably approximate more closely to what humans receive naturally, it is recommended that they should be used for future testing of all potential vaccines in baboons.

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.

Variable species and stage specificity of schistosomulum surface epitopes recognized by mice vaccinated with highly irradiated cercariae

Antibodies from mice vaccinated with highly irradiated Schistosoma mansoni or S. haematobium cercariae were used to characterize schistosomulum surface epitopes which were found to be diverse in their species and stage specificities. The epitopes recognized on the Mr greater than 200,000 and 15,000 schistosomulum surface antigens of S. mansoni and the Mr greater than 200,000 schistosomulum surface antigen of S. haematobium were found to be cross-specific whereas those on the Mr 38,000, 32,000 and 20,000 schistosomulum surface antigens of S. mansoni and the Mr 35,000, 30,000 and 24,000 schistosomulum surface antigens of S. haematobium were only immunoprecipitated by homologous antibody and are thus possible targets of the protective species-specific immunity stimulated by highly irradiated cercariae. The epitopes recognized on the Mr greater than 200,000 and 38,000 antigens of S. mansoni were shown to cross-react with both the egg and the adult worm whereas those on the Mr 32,000 and 20,000 antigens only cross-reacted with the adult worm, and those on the Mr 15,000 antigen cross-reacted with neither the adult worm nor the egg. In addition the epitopes on the Mr 38,000 and 32,000 antigens were demonstrated to be polypeptide in nature. Those on the Mr greater than 200,000, 20,000 and 15,000 antigens, on the other hand, could not be conclusively defined.

Protective monoclonal antibodies from mice vaccinated or chronically infected with Schistosoma mansoni that recognize the same antigens.

An IgM monoclonal antibody, designated mAb 1.G1, has been generated from spleen cells of mice immunized with irradiated Schistosoma mansoni cercariae. As determined by indirect immunofluorescence, mAb 1.G1 binds to the surface membrane of schistosomula and to the ciliated plates of miracidia. mAb 1.G1 also binds to the protonephridial systems of live adult worms and denuded, acetone-fixed schistosomula. Western blot analysis shows that the target epitope of this mAb is found on Nonidet P-40-solubilized schistosomular antigens ranging in molecular size from 85 to 130 kDa and ciliated plate antigens of miracidia at 92, 95, and 102 kDa. The recognized epitope in an 8 M urea adult worm extract is found on a 97-kDa molecule. In addition, mAb 1.G1 mediates a high level of complement-dependent cytotoxic activity against schistosomula when used in an in vitro assay. In passive immunization experiments, approximately 40% protection was provided mice when mAb 1.G1 was administered either at the time of challenge or when given 8 days postchallenge. However, when administered 15 days postchallenge, mAb 1.G1 failed to mediate passive protection. The ability of mAb 1.G1 to mediate protection in vivo correlates with its recognition of epitopes on the surfaces of live schistosomula up to 8 days but not at 15 days. Western blot analysis showed that the antigens were contained within Nonidet P-40 extracts of schistosomula during the same time period. Furthermore, a second monoclonal antibody (mAb 4.4B) derived from mice chronically infected with S. mansoni exhibits the identical properties as described for mAb 1.G1.

In vivo T cell depletion regulates resistance and morbidity in murine schistosomiasis.

These studies assessed the roles of subpopulations of T lymphocytes in inducing and modulating resistance to schistosomiasis and thereby influencing subsequent morbidity. C57BL/6 mice were depleted in vivo of Lyt-1+, Lyt-2+, and L3T4+ cells by the daily administration of monoclonal antibodies. The development of protective immunity, induced by exposure to irradiated Schistosoma mansoni cercariae as expressed in depleted animals, was compared to that demonstrated in undepleted, normal, and congenitally athymic C57BL/6 mice. The development of morbidity was determined by spleen weight, portal pressure and reticuloendothelial system activity. The results indicated that depletion of specific subpopulations of T lymphocytes minimally affected the primary development of parasites; however, depletion strongly influenced the development of resistance to the parasite and subsequent morbidity due to infection. Depletion of T lymphocytes by anti-Lyt-1+ or anti-L3T4+ antibody decreased the development of resistance, antibody and delayed-type hypersensitivity directed against schistosome antigens. Morbidity due to disease was increased. Depletion of Lyt-2+ cells produced opposite changes with augmented resistance and reduced morbidity. Congenitally athymic mice developed minimal resistance and morbidity. Moreover, resistance was inversely related to the morbidity shown by a given animal. These studies indicate that the development of protective immunity to S. mansoni cercariae is regulated by discrete subpopulations of T lymphocytes. The feasibility of decreasing morbidity by increasing specific immunologically mediated resistance is suggested.

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.

Effects of immunomanipulations on resistance induced by irradiated Schistosoma mansoni cercarial sensitization of C57BL/6 and CBA/J mice.

Mice sensitized with radiation-attenuated Schistosoma mansoni cercariae were treated by a variety of procedures known to interrupt immunoregulatory circuitries in attempts to alter the resistance induced by irradiated cercarial sensitization. Both highly resistant C57BL/6 and the often poorly resistant CBA/J strains were examined. Immunomanipulations included adult thymectomy, or administration of different drugs in relation to the time of challenge infection (day 0). Adult thymectomy was performed 3 weeks prior to sensitization or 3 weeks prior to challenge. Drug treatment included cyclophosphamide given at a dose of 20 mg/kg on alternate days, day -11 through day +11, cimetidine at 50 mg/kg/day, day -7 through day +21, or indomethacin at 2.5 mg/kg/day, day 0 through day +10. In most experiments, irradiated cercarial sensitized C57BL/6 mice developed significant resistance which was not altered by the immunomanipulative regimens used. If however, as fortuitously occurred in two adult thymectomy experiments, irradiated cercarial sensitization did not induce significant resistance, immunomanipulation by adult thymectomy allowed the development of a significant protected state. Similarly, adult thymectomy or cimetidine treatment in concert with immunization of CBA/J mice conferred moderate, statistically significant levels of stable resistance in this normally "less resistant" strain.

Cutaneous sensitivity induced by immunization with irradiated Schistosoma mansoni cercariae : II. Autoimmunoregulation of cell-mediated cutaneous sensitivity and its reversal

C57BL/6 mice sensitized by abdominal, dermal exposure to irradiated (50 kR) Schistosoma mansoni cercariae develop partial protection against subsequent exposure with unattenuated cercariae and express cell-mediated cutaneous sensitivity upon challenge with irradiated cercariae. Autoimmunoregulation occurs as a part of this sensitization, and can be demonstrated by augmentation of cutaneous sensitivity upon use of appropriate regimens of cyclophosphamide. Mice exposed to irradiated cercariae by either intraperitoneal or ear pinna routes developed a transient hyporesponsiveness to cercarial challenge. This unresponsiveness was also reversed by pretreatment with cyclophosphamide. Timed removal of the site of effective sensitization (abdominal skin) 7 days after exposure consistently led to reduced cutaneous responsiveness. This artificially induced hyporesponsiveness was reversed by either cyclophosphamide treatment or systemic administration of anti-I-J b, but not anti-I-J k sera. The data indicate the involvement of cyclophosphamide-sensitive, I-J-bearing T-suppressor cells or factors in the autoimmunoregulation that controls this cutaneous sensitivity. Parallel challenge infection studies in immunized mice treated with cyclophosphamide demonstrated that the resultant augmentation of cutaneous sensitivity did not lead to concomitantly elevated levels of resistance. Furthermore, successful adoptive cell transfer of cutaneous responsiveness also did not ensure protection against cercarial challenge. These observations indicate that dermal cell-mediated anti-cercarial responsiveness is not a sufficient mechanism to explain resistance in mice immunized with irradiated cercariae.

Cutaneous sensitivity induced by immunization with irradiated Schistosoma mansoni cercariae : I. Induction, elicitation, and adoptive transfer analysis of cell-mediated cutaneous sensitivity

Exposure of C57BL/6 mice to highly irradiated (50 kR) cercariae of Schistosoma mansoni leads to the development of partial resistance against subsequent challenge with unattenuated cercariae. We have analyzed the cellular immune responses that occur during the afferent and efferent phases of this protective sensitization. Mice were immunized by exposure to irradiated S. mansoni cercariae. After challenge with irradiated cercariae, delayed-type (18–72 hr) cutaneous sensitivity reaction sites were rich in mononuclear cells and eosinophils. This reactivity was established by 4 days after sensitization, reached its maximum between 7 and 14 days after sensitization, and was maintained for over 20 weeks. These challenge reactions could be abrogated by treatment with either 200 mg/kg cyclophosphamide or 5 mg of hydrocortisone. Syngeneic adoptive transfer of cutaneous sensitivity was accomplished with lymphoid cells from the draining lymph nodes or spleens of mice sensitized 7–14 days previously. Negative selection studies of nylonwool non-adherent cells from sensitized donors demonstrated that the cells responsible for transferring this eosinophil-rich, delayed-type cutaneous sensitivity to S. mansoni irradiated cercariae were Thy-1+, Lyt 1+, Lyt 2−, surface Ig- lymphocytes.

Evidence against the existence of specific Schistosoma mansoni subpopulations which are resistant to irradiated vaccine-induced immunity.

When mice are immunized with irradiated Schistosoma mansoni cercariae a proportion of the subsequent cercarial challenge always escapes killing and matures to egg-laying adults. This report investigates the possibility that incomplete immunity in this system is governed by a genetically-determined insusceptibility of a particular schistosome subpopulation. To do this we tested whether more immunoresistant schistosomes would develop following successive passages of progeny of the resistant worms through immunized mice. Mice were immunized with 500 50 Krad-irradiated cercariae, and challenged with normal cercariae when immunity was at its peak. After five successive passages through snails and immune mice, progeny of those parasites which escaped immune killing were no more refractory to vaccine-induced resistance than the original stock maintained in nonimmune mice. Additionally, the "passaged" isolates did not differ from the original stock in their ability to induce protection following irradiation. Our results indicate that with this model of acquired resistance incomplete immunity is unlikely to be due to a subpopulation of the parasites possessing a genetically-determined insusceptibility to killing.

Economic evaluation of the production impact of bovine schistosomiasis and vaccination in the Sudan

This study was done to evaluate the estimated economic consequences of the recent discovery that an irradiated Schistosoma bovis vaccine was effective in reducing mortality and intensity of infection in cattle after field exposure to S. bovis in the White Nile province. The benefits and costs were hypothesized to occur over a 5-year period starting after the vaccine had been further developed to optimal commercial usefulness. The potential benefits of vaccination are from the avoidance of mortality and growth delay losses caused by S. bovis infection and were based on an owner survey conducted in 1981. These benefits were discounted from the time of their potential marketing opportunity to the first year of a vaccination program at 15% per annum and were valued on a basis of 1982 prices for live cattle exported to Egypt and Saudi Arabia. Variations in benefits stem from degree of infection probability, mortality and morbidity estimates and percent of animals vaccinated. Since clinical schistosomiasis (“gorag” — sunken-eyed appearance) and associated production losses occur almost exclusively in 6- to 30-month-old cattle, and there is evidence for long-term immunity, vaccinations would be given to cattle in this age-specific group or younger once in their lifetime. The principal variation in vaccination program costs, also valued at 1982 prices, is from vaccine production costs; $0.50 or $4.00 per dose. A vaccine efficacy of 70%, observed in a previously reported field trial, was used in these calculations. Present value benefit—cost (b–c) ratios were estimated for the central, western and southern areas for high- and low-level effects of S. bovis impact on production and the vaccination program, cost and effectiveness. In an area (central provinces) of high infection probability (90%), high percentage of animals vaccinated (90%), high mortality (7.1%), and low vaccine production costs, the b–c ratio was 12.7. In contrast, a b–c ratio of 0.7 was estimated for an area (southern provinces) assuming low infection probability (50%), low percent of animals vaccinated (50%), lower mortality (3.55%) and high vaccine production costs. Potential returns from increased future milk and calf production and from faster herd build-up with younger females were not included in these benefit calculations. These results indicate that under most conditions further development of the vaccine and cost-effective vaccine production techniques would yield very favorable returns from improved livestock production efficiency in the Sudan. Export prices were assumed to not vary significantly with increased supply of export-quality cattle resulting from the estimated production losses avoided by vaccination against schistosomiasis.

Lack of resistance to Schistosoma japonicum in mice immunized with irradiated S. mansoni cercariae

Mice immunized with irradiated Schistosoma mansoni cercariae were resistant to challenge with S. mansoni cercariae (mean resistance 53%) but not to challenge with S. japonicum cercariae (mean resistance −5%). Furthermore, the antibodies induced by vaccination with irradiated S. mansoni cercariae were more reactive with S. mansoni than with S. japonicum schistosomula. These results support the concept that the resistance induced by vaccination with irradiated cercariae is immunologically specific.

Resistance induced by normal and irradiated Schistosoma mansoni: ability of various worm stages to serve as inducers and targets in mice.

Lung stage schistosomula exposed to 50 kilorads of gamma irradiation induced significant resistance to challenge infection with Schistosoma mansoni following intravenous (tail or mesenteric vein), intramuscular, or intraperitoneal injection into mice. Similar or higher levels were induced with irradiated cercariae, while irradiated 3- or 4-week-old worms induced little resistance. Non-irradiated day 6 and day 12 lung schistosomula injected into mice immunized with irradiated cercariae were susceptible to elimination, though to a lesser extent than a challenge infection administered at the cercarial stage. Day 20 liver worms injected into a mesenteric vein were not susceptible to irradiated cercaria-induced resistance. In contrast, cercariae, day 6 lung schistosomula, day 12 lung schistosomula and day 20 liver worms were all susceptible to the resistance induced by a chronic (non-irradiated) infection.