Lung-stage expression of a major schistosome surface antigen

The topographical expression of a glycoprotein of 180,000 molecular weight on the surface of lung-stage Schistosoma mansoni schistosomula was determined by immunofluorescence microscopy using a monoclonal antibody. Postfixation treatment with graded ethanols enhanced specific immunofluorescent staining of adult worms, and was required for detection of the antigen on the surfaces of lung-stage schistosomula. The epitope recognized by the monoclonal antibody was also present on the surfaces of adult Schistosoma haematobium, but not on those of Schistosoma japonicum.     

Apoptosis governs the elimination of Schistosoma japonicum from the non-permissive host Microtus fortis

The reed vole, Microtus fortis, is the only known mammalian host in which schistosomes of Schistosoma japonicum are unable to mature and cause significant pathogenesis. However, little is known about how Schistosoma japonicum maturation (and, therefore, the development of schistosomiasis) is prevented in M. fortis. In the present study, the ultrastructure of 10 days post infection schistosomula from BALB/c mice and M. fortis were first compared using scanning electron microscopy and transmission electron microscopy. Electron microscopic investigations showed growth retardation and ultrastructural differences in the tegument and sub-tegumental tissues as well as in the parenchymal cells of schistosomula from M. fortis compared with those in BALB/c mice. Then, microarray analysis revealed significant differential expression between the schistosomula from the two rodents, with 3,293 down-regulated (by ≥ 2-fold) and 71 up-regulated (≥ 2 fold) genes in schistosomula from the former. The up-regulated genes included a proliferation-related gene encoding granulin (Grn) and tropomyosin. Genes that were down-regulated in schistosomula from M. fortis included apoptosis-inhibited genes encoding a baculoviral IAP repeat-containing protein (SjIAP) and cytokine-induced apoptosis inhibitor (SjCIAP), genes encoding molecules involved in insulin metabolism, long-chain fatty acid metabolism, signal transduction, the transforming growth factor (TGF) pathway, the Wnt pathway and in development. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) and PI/Annexin V-FITC assays, caspase 3/7 activity analysis, and flow cytometry revealed that the percentages of early apoptotic and late apoptotic and/or necrotic cells, as well as the level of caspase activity, in schistosomula from M. fortis were all significantly higher than in those from BALB/c mice.     

Innate immunogenicity and in vitro protective potential of Schistosoma mansoni lung schistosomula excretory–secretory candidate vaccine antigens

Excretory–secretory products (ESP) of Schistosoma mansoni developing larvae are ideal potential vaccines as such molecules may readily induce host primary immune responses, and local memory immune response effectors that would target, surround, and pursue the larvae while negotiating the lung blood capillaries. We herein characterized the cytokines response ESP, e.g., SG3PDH, 14-3-3-like protein, TPX, and calpain induce in the natural context of infection, and defined the global cytokine profile conducive to effective schistosome larvae killing. Accordingly, spleen cells (SC) taken from naïve, and 7-, or 9-day S. mansoni-infected mice were stimulated in vitro with the selected ESP, in a recombinant or multiple antigen peptide (MAP) form, and examined for production of T helper type (Th) 1, Th2, and Th17 cytokines, and the ability to mediate in vitro attrition of lung-stage schistosomula. The study indicated that larval ESP principally elicit Th1 and Th17 type cytokines. Recombinant SG3PDH was the only test ESP to additionally activate SC from S. mansoni-infected BALB/c mice to release higher IL-4 levels than unstimulated SC and mediate significant (P < 0.0001) in vitro attrition of lung-stage larvae. Thus, our data suggested that a balance between Th1, Th17, and Th2 cytokines is required for effective schistosome larval elimination.     

Vaccination against Schistosomiasis: The case for Lung-stage Antigens

The development of an effective vaccine against human schistosomiasis remains a highly desirable yet elusive goal. In this article, Adrian Mountford and Richard Harrop focus attention on an approach that aims to identify proteins from Schistosoma mansoni that are capable of stimulating protective Th1 cell-mediated immune responses. They propose that the most likely source of such antigens is the lung-stage schistosomulum.     

Identification of immunodominant Th1-type T cell epitopes from Schistosoma japonicum 28 kDa glutathione-S-transferase, a vaccine candidate

Th1-type cytokines produced by the stimulation of Th 1-type epitopes derived from defined schistosome-associated antigens are correlated with the development of resistance to the parasite infection. Schistosoma mansoni 28 kDa glutathione-S-transferase （Sm28GST）, a major detoxification enzyme, has been recognized as a vaccine candidate and a phase II clinical trial has been carried out. Sheep immunized with recombinant Schistosoma japonicum 28GST （Sj28GST） have shown immune protection against the parasite infection. In the present study, six candidate peptides （P1, P2, P3, P4, P7 and P8） from Sj28GST were predicted, using software, to be T cell epitopes, and peptides P5 and P6 were designed by extending five amino acids at the N-terminal and C-terminal of P1, respectively. The peptide 190-211 aa in Sj28GST corresponding to the Th1-type epitope （190-211 aa） identified from Sm28GST was selected and named P9. The nine candidate peptides were synthesized or produced as the fusion protein with thioredoxin in the pET32c（＋）/BL21（DE3） system. Their capacity to induce a Th1-type response in vitro was measured using lymphocyte proliferation, cytokine detection experiments and flow cytometry. The results showed that P6 （73-86 aa） generated the strongest stimulation effect on T cells among the nine candidate peptides, and drove the highest level of IFN-γ, and IL-2. Therefore, P6 is a functional Thl-type T cell epitope that is different from that in Sm28GST, and will be useful for the development of effective vaccines which can trigger acquired immunity against S. japonicum. Moreover, our strategy of identifying the Thl-type epitope by a combination of software prediction and experimental confirmation provides a convenient and cost-saving alternative approach to previous methods.     

IgM antibodies recognizing carbohydrate epitopes shared between schistosomula and miracidia of Schistosoma mansoni that block in vitro killing

A series of monoclonal antibodies (mAb) was raised in mice against Schistosoma mansoni, which recognized a carbohydrate determinant on a major Mr greater than 200,000 schistosomulum surface antigen. These mAb cross-reacted with the surface of cercariae and miracidia and with schistosomula of S. haematobium and S. bovis. Other mAb were generated that only recognized a Mr 20,000 schistosomulum surface antigen; they did not cross-react with eggs or miracidia and were species specific. The anti-Mr 20,000 mAb of the IgG1 isotype exhibited high levels of complement-dependent cytotoxicity to schistosomula in vitro. IgM mAb that recognized carbohydrate epitopes of the Mr greater than 200,000 surface antigen blocked the lethal activity of the anti-Mr 20,000 mAb. The IgM anti-Mr greater than 200,000 mAb also reduced complement-dependent cytotoxicity of serum from mice vaccinated with irradiated cercariae.     

Depletion of Schistosoma mansoni lung-stage schistosomula cholesterol by methyl-beta-cyclodextrin dramatically increases specific antibody binding to surface membrane antigens

Schistosoma mansoni lung-stage larvae appear to not bind antibodies from radiation vaccine or infection sera in the membrane immunofluorescence test. However, treatment of ex vivo lung-stage schistosomula with methyl-beta-cyclodextrin, a hydrophobic oligosaccharide that specifically extracts cholesterol from plasma membranes, induced readily detectable binding of specific antibodies in a concentration- and time-dependent manner. Surface membrane antigen binding of specific antibodies was also conclusively demonstrated by quantitative absorption of anti-schistosome sera with intact ex vivo larvae. These data together suggest that confinement of lung-stage schistosomula surface membrane antigens in cholesterol-rich sites allows only monovalent antibody binding, which can be detected by absorption and not by direct serology.     

Schistosomajaponicum： Isolation and Identification of Peptides Mimicking Ferritin Epitopes from Phage Display Library

Although schistosomicidal drug and other control measures (including hygiene and snail control) are availableschistosomiasis continues to afflict an estimated 200 million and kill 20 thousand people every year, new approachefor controlling this disease are urgently needed. Cocktail vaccine, such as multiple antigen peptidvaccine, emerged as the most potentially powerful meansFor multiple antigen peptide vaccine that bases on domainor subdomains of antigens, the key is to obtain antigeniepito…     

A purified 28,000 dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental schistosomiasis

We have purified a 28,000 dalton (P28) protein from Schistosoma mansoni adult worms and used it to immunize Fischer rats. Immunofluorescence assays demonstrated that the P28 antigen was mainly located in the parenchyma of the schistosomulum and of the adult worm, including the dorsal spines of the parasite. Western blot analysis revealed that this antigen was present in three species of schistosomes: S. mansoni, S. japonicum, and S. bovis. The antibody response raised against this protein was able to kill S. mansoni schistosomula in in vitro cytotoxicity assays in the presence of rat eosinophils. The inhibition of this cytotoxic activity by an aggregated myeloma IgG2a indicated that one of the major isotypes involved in this in vitro model is IgG2a. The passive transfer of P28 antisera induced a significant level of protection against experimental infection. Moreover, we have immunized Fischer rats and BALB/c mice with the purified 28,000 dalton protein and observed a marked decrease (up to 70%) in the parasite burden in both experimental infection models.     

Equilibrium in lung schistosomula sphingomyelin breakdown and biosynthesis allows very small molecules, but not antibody, to access proteins at the host-parasite interface

The mechanism by which lung-stage schistosomula expose proteins at the host-parasite interface to nutrient, but not antibody, uptake has been obscure. We have found that Schistosoma mansoni and Schistosoma haematobium larvae emerging from host lung at a pH of around 7.5, and fixed with diluted formaldehyde (HCHO), readily bind specific antibodies in indirect membrane immunofluorescence. Data on inhibitors and activators of parasite tegument-bound, magnesium-dependent, neutral sphingomyelinase (nSMase), and sphingomyelin biosynthesis inhibitors revealed that equilibrium in schistosomular sphingomyelin breakdown and biosynthesis prevents antibody binding, yet permits access of small HO-CH2-OH polymers to interact with and cross-link proteins at the host-parasite interface, allowing for their serological visualization.     

Antibody-dependent murine macrophage-mediated damage to Schistosoma mansoni schistosomula in vitro

Antibody-dependent cell-mediated cytotoxicity (ADCC) against schistosomula of the human parasite Schistosoma mansoni was demonstrated using antisera from mice plus peritoneal exudate cells (PEC). PEC were divided into plastic-adherent (96% macrophages, 4% lymphocytes) and nonadherent (92% lymphocytes, 8% macrophages) cell populations. Four criteria of ADCC were used, including minimal and maximal cell attachment, and death of and uptake of trypan blue by schistosomula. Using cells from normal mice and antisera from schistosome-infected mice, macrophages adhered to, damaged the tegument and underlying structures of, and killed schistosomula when observed following 18 hr incubation. In homologous systems, the results were similar when outbred CD-1 and inbred BALB/c mice were compared, except that potency of antisera from the latter mice decreased after 6–7 weeks postinfection, whereas the opposite was true for the former strain of mice. Nonadherent cells also exhibited ADCC against schistosomula, but the potency was considerably lower than that of adherent cells. These complement-independent ADCC reactions were stage-specific for the schistosomulum in that no reactions occurred with adult worms.     

The modulation of expression of polypeptide surface antigens on developing schistosomula of Schistosoma mansoni

Five low m.w. polypeptide antigens are expressed on the surface of freshly transformed schistosomula of Schistosoma mansoni, and were reproducibly identified by surface labeling with 125I by using IODOGEN and immunoprecipitating with immune mouse sera. These molecules have approximate m.w. of 38,000, 32,000, 20,000, 17,000, and 15,000. They correspond to antigens recognized previously by lactoperoxidase-catalyzed iodination. Analysis of the surface of developing schistosomulum demonstrated that the 38,000 and 17,000 dalton antigens were lost from the parasite surface during 48 hr of in vitro culture. This process was not dependent on the presence of host serum. The two antigens were not lost due to shedding into the culture medium but were apparently sequestered to a site where they were no longer available for surface labeling. The 32,000, 20,000, and 15,000 dalton antigens, however, remained exposed on the schistosomulum surface for up to 2 days of in vitro culture. The expression of two new antigens was also induced by culture in vitro: a doublet of approximately 45,000 daltons and an antigen of approximately 11,000 daltons. The expression of the former was dependent on the presence of serum. These results demonstrate that the development of the schistosomula surface is a complex process, with events both dependent and independent of the presence of serum. In addition, the expression of polypeptide antigens is not coordinated, and antigens are lost, retained, or appear on the schistosomulum surface during the early stages of maturation.     

Three major surface antigens of Schistosoma mansoni are linked to the membrane by glycosylphosphatidylinositol

Schistosomula of Schistosoma mansoni were examined for the presence of glycosylphosphatidylinositol (GPI) anchored surface membrane Ag. Parasites were surface iodinated and cultured in the presence or absence of a crude phospholipase C (PLC) preparation or phosphatidylinositol-specific PLC (PIPLC). Culture supernatants were then analyzed: 1) by centrifugation to ascertain which molecules released from the surface were soluble or contained in membrane vesicles; 2) by immunoprecipitation with antibodies specific for the "cross-reacting determinant," an epitope revealed on some GPI-anchored proteins only after cleavage of the diacylglycerol from the protein by PIPLC, and 3) by immunoprecipitation with immune mouse sera to establish co-identity with previously described, immunologically relevant surface Ag. By using these techniques, schistosomula were shown to possess three GPI-anchored surface Ag of m.w. 38,000, 32,000 and 18,000 which are spontaneously released from the surface of schistosomula in association with membrane, but remain insoluble until cleaved by PIPLC. All three molecules were recognized by antibodies from mice vaccinated with irradiated cercariae and/or chronically infected mice. Moreover, the m.w. 38,000 component was recognized by a previously described protective mAb (E.1). A major developmental modification appears to occur in the expression of these molecules because, by the same techniques, no GPI-anchored surface Ag were detectable on 7-day-old lung stage parasites. The finding that these important parasite immunogens are GPI-anchored and released from the surface of the parasite in membrane vesicles may, in part, explain why they elicit strong immune responses capable of damaging the schistosomulum tegument.     

Failure of anti-Fasciola antisera to induce antibody-dependent, eosinophil-mediated killing of Schistosoma mansoni schistosomula

Sera from rabbits or humans infected with Fasciola hepatica were tested for their ability to kill Schistosoma mansoni schistosomula in an antibody-dependent, eosinophil-mediated in vitro assay. In addition, anti-F. hepatica antisera raised in rabbits or calves, including one to a Fasciola/Schistosoma cross-reactive, cross-protective defined immunity antigen, were also tested for their killing ability. None of these antisera induced damage to S. mansoni schistosomula in vitro, even when enhanced with mononuclear cell supernatants containing eosinophil-activating factor. Serum from humans with S. mansoni did induce schistosomulum killing in vitro when tested under these same conditions. These results suggest that the mechanism of immunity to schistosomes induced by Fasciola antigens at the level of the schistosomula is mediated by factors other than eosinophils.     

日本血吸虫尾蚴及童虫的生理学比较

本文介绍了经0.05％中性红-氢氧化钾染色确定为活的血吸虫尾蚴和童虫的生理学比较结果。与尾蚴相比,童虫体表对PAS及Alcian蓝阳性反应消失,与抗血清接触不再产生套膜反应;对水不能耐受,在淡水中6小时内已全部死亡,而在生理盐水中仍有92.7％存活;钻腺内含物排空,童虫不能再经皮肤感染,但越过皮肤屏障若被注入皮下或腹腔则分别有45.4—46.5％及56.9—64.9％发育为成虫。     

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.     

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats

Antibodies from Schistosoma mansoni-infected rats, unlike mice, show a higher titer for schistosome apical tegumental antigens compared with non-apical membrane antigens. These antibodies bind to the surface of living lung-stage worms and to formaldehyde-fixed adult worms. We produced a single-chain antibody Fv domain (scFv) phage library displaying the antibody repertoire of rats highly immune to schistosome infection and we selected for scFvs that recognize the host-exposed surface of worms. Five unique rat scFvs (Teg1, Teg4, Teg5, Teg20 and Teg37) were obtained which recognize schistosome surface epitopes. Each of the scFvs recognizes the surface of living schistosomula and lung-stage schistosomules and/or the surface of formaldehyde-fixed adult worms. None of these scFvs reproducibly stained living adult worms. This suggests that a change occurs during the transition from lung schistosomules to 4-week adults such that at least some surface antigens, although remaining on the surface in living adult worms, can no longer be immunologically stained. Teg1 and Teg4 scFvs both recognize specific bands on Western blots. No bands were observed for the other three scFvs, suggesting that these scFvs may recognize non-protein or conformationally-dependent epitopes. Teg1 was unambiguously identified as recognizing the S. mansoni tetraspanin antigen, SmTSP-2, within the large extracellular domain. Teg4 recognizes a 35 kDa band tentatively identified as Sm29 by proteomic analysis. These scFvs can now be used to characterize schistosome epitopes at the host-parasite interface, to target worms in vivo, and to study the mechanisms by which these worms naturally evade immune damage to the tegument within permissive hosts.