Schistosoma mansoni Annexin 2: Molecular characterization and immunolocalization

We here describe the cloning and characterization of the Schistosoma mansoni Annexin 2, previously identified in the tegument by proteomic studies, and as an up-regulated gene in schistosomulum stage by microarray data. In silico analysis predicts a conserved core containing four repeat domains of Annexin (ANX) and a variable N-terminal region similar to that described for mammalian isoforms. Real-time RT-PCR and Western blot analysis determined that S. mansoni Annexin 2 is significantly up-regulated in the transition from free-living cercaria to schistosomulum and adult worm parasitic stages. Immunolocalization experiments and tegument membrane preparations confirmed Annexin 2 as a protein mainly localized in the tegument of schistosomula and adult worms. Furthermore, it binds to the tegument surface membranes in a calcium-dependent manner. These results suggest that S. mansoni Annexin 2 is closely associated to the tegument arrangement, being a potential target for immune intervention.     

Surface antigens on cercariae,schistosomula and adult worms of Schistosoma mansoni

Shah J. and Ramasamy R. 1982. Surface antigens on cercariae, schistosomula and adult worms of Schistosoma mansoni. International Journal for Parasitology12: 451–461. The surface protein antigens of Schistosoma mansoni were radiolabelled by lactoperoxidase catalysed I¹²⁵-iodination and analysed by immune-precipitation and polyacrylamide gel electrophoresis. The results showed that regularly labelled surface antigens of mol. wts >150,000, 78,000, 45,000 and 22,000 were present on adult worms. Common surface antigens were observed on the cercariae, schistosomula and adult worms. It is suggested that surface antigens released from living adult worms can sensitise a host to react against the invading schistosomula of a secondary infection. However, the failure to vaccinate mice using material containing adult worm surface antigens suggests that the induction of protective immunity is a complex phenomenon.     

Schistosoma japonicum: Tsunagi/Y14 protein plays a critical role in the development of the reproductive organs and eggs

Tsunagi/Y14 is an evolutionarily conserved RNA-binding protein that is required for the maintenance of oogenesis and the masculinization of the germ-line in many animal models. We speculated that Tsunagi/Y14 might also regulate reproductive organ development in Schistosoma japonicum (S. japonicum, Sj). Sj Tsunagi/Y14 and control double-stranded RNAs were introduced into schistosomula by electroporation respectively. These transfected schistosomula were cultured in vitro for 1, 3 or 5 days. The mRNA and protein levels of the target gene in the cultured schistosomula were significantly suppressed compared with those of the control group. Furthermore, BALB/c mice were infected with the transfected schistosomula for 6 weeks and were sacrificed to harvest the adult worms. We found that the silencing of Sj Tsunagi/Y14 led to defects in reproductive organs development in both male and female worms. Moreover, it also affected the size, quantity and activity of the eggs in the mice liver. Our findings indicated that Tsunagi/Y14 plays a critical role in the development of reproductive organs and eggs in S. japonicum.     

Schistosoma mansoni tegument protein Sm29 is able to induce a Th1-type of immune response and protection against parasite infection

Background

Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate.

Methods and Findings

We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p<0.05). Protective immunity in mice was associated with high titers of specific anti-Sm29 IgG1 and IgG2a and elevated production of IFN-γ, TNF-α and IL-12, a typical Th1 response. Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to worms from control mice revealed a significant (q<0.01) down-regulation of 495 genes and up-regulation of only 22 genes. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals, suggesting that under immune attack schistosomes reduce the expression of critical surface proteins.

Conclusion

This study demonstrates that Sm29 surface protein is a new vaccine candidate against schistosomiasis and suggests that Sm29 vaccination associated with other protective critical surface antigens is the next logical strategy for improving protection.     

Schistosoma mansoni: immunization of cynomolgus monkeys by injection of irradiated schistosomula.

Although the immunization of primates with irradiated schistosome cercariae has been demonstrated, no success has been reported by injection with the irradiated schistosomule stage. The present investigation was designed to test whether cynomolgus monkeys could be protectively immunized with ⁶⁰Co-irradiated Schistosoma mansoni schistosomula. Monkeys injected once with 10⁴ irradiated schistosomula (50 krad at 4 krad/min) had 52% fewer challenge worms than the control group at necropsy. Four immunizations did not induce a higher level of resistance. At 50 days post-challenge, the immunized monkeys excreted 80% fewer eggs than did the control animals. An attempt to enhance irradiated schistosomule-induced protection with tetramisole · HCl was unsuccessful.     

Schistosoma japonicum: Efficient and rapid purification of the tetraspanin extracellular loop 2, a potential protective antigen against schistosomiasis in mammalian

The extracellular loop 2 of a tetraspanin from Schistosoma japonicum (Sj-TSP-2) is homologous to Schistosoma mansoni TSP-2. In our initial study, Sj-TSP-2 is an identical antigen against schistosomiasis caused by S. japonicum. Through the pET32 vector system and nickel (Ni)-absorbed chelating Sepharose, Sj-TSP-2 was expressed and purified as a soluble fusion constructed with an N-terminal thioredoxin-His₆-EK protease site tag (Trx-TSP-2). In phosphate buffer (PB) with a low concentration of imidazole, the Trx-TSP-2 fusion protein was efficiently cleaved by enterokinase (EK). Sj-TSP-2 was isolated and enriched using cobalt (Co)-absorbed chelating Sepharose and HiTrap SP column. Character of the protein was analyzed via animal experiments and then clinical trials. The purification approach yielded pure Sj-TSP-2, which will provide feasible advices for discovering vaccines against schistosomiasis.     

β-Lapachone: A naphthoquinone with promising antischistosomal properties in mice

The activity of β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione, β-lap) against different stages of Schistosoma mansoni was investigated in mice. Mice infected with 50 cercariae (BH strain) were intraperitoneally treated at a dose of 50 mg/kg for 5 consecutive days, starting on the 1st, 14th, 28th and 45th days after infection, to evaluate the effect of β-lap on skin schistosomula, lung schistosomula, young worms (before oviposition) and adult worms (after oviposition), respectively. All animals were euthanized 60 days after infection. β-Lap significantly reduced (p < 0.001) the number of worms in 29.78%, 37.2%, 24.2% and 40.22% when administered during the phases of skin schistosomula, lung schistosomula, young worms and adult worms, respectively. Significant reduction was also achieved in terms of female burden. In all groups, there was significant reduction in the number of eggs and granulomas in the hepatic tissue. When the intervention was performed during the phase of adult worms, β-lap reduced the size of hepatic granulomas and changed the oogram pattern, lowering the percentage of immature eggs and increasing the percentage of mature and dead eggs. Our data indicate that β-lap has moderate antischistosomal properties. Its molecule may also be used as a prototype for synthesis of new naphthoquinone derivatives with potential schistosomicidal properties. Further studies with different formulations containing β-lap are needed to clearly establish the best dose and route of administration and its mechanism of action against schistosomes.     

Schistosoma mansoni: topochemical features of cercariae, schistosomula, and adults

The teguments of developing and mature cercariae, recently transformed, and 1-wk-old schistosomula and adult worms were examined for the ultrastructural location of macromolecular carbohydrates and polyelectrolytes. The surface of mature cercariae within sporocysts and cercariae released from the snail is covered by a filamentous coat which reacts with cytochemical reagents for the demonstration of vicinal glycols, but neither the coat nor the surface of the tegument plasmalemma binds cationic colloidal iron at low pH.Upon penetrating mammalian skin, the cercaria sheds its surface coat; the tegument surface of newly transformed schistosomula, older schistosomula and adult worms stains en bloc with acidic colloidal iron, as does the tegument plasmalemma of mature cercariae if the overlying filamentous coat is first removed by physicochemical means. The cercarial coat thus serves to mask anionic groups at the surface of the tegument plasmalemma which become functionally exposed after penetration of the mammalian host. The distribution of colloidal iron binding sites coincides with those for the carbohydrate-complexing phytohemmagglutnin, concanavalin A, which suggests that these membrane-fixed anions are acid mucopolysaccharides, glycoproteins or glycolipids. Carbohydrate-containing material was also localized within membrane-bound vesicles of the tegument matrix and perikarya of developing cercariae and postcercarial schistosomes, suggesting that surface mucosubstances contributing to the tegument glycocalyx of these worms are elaborated, at least in part, by the tegument itself.     

Schistosome Syntenin Partially Protects Vaccinated Mice against Schistosoma mansoni Infection

Background

Schistosomiasis is a neglected tropical disease caused by several species of trematode of the genus Schistosoma. The disease affects more than 200 million people in the world and causes up to 280,000 deaths per year, besides having high morbidity due to chronic illness that damages internal organs. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Among the most promising molecules as vaccine candidates are the proteins present in the tegument and digestive tract of the parasite.

Methodology/Principal Findings

In this study, we describe for the first time Schistosoma mansoni syntenin (SmSynt) and we evaluate its potential as a recombinant vaccine. We demonstrate by real-time PCR that syntenin is mainly expressed in intravascular life stages (schistosomula and adult worms) of the parasite life cycle and, by confocal microscopy, we localize it in digestive epithelia in adult worms and schistosomula. Administration of siRNAs targeting SmSynt leads to the knock-down of syntenin gene and protein levels, but this has no demonstrable impact on parasite morphology or viability, suggesting that high SmSynt gene expression is not essential for the parasites in vitro. Mice immunization with rSmSynt, formulated with Freund''s adjuvant, induces a Th1-type response, as suggested by the production of IFN-γ and TNF-α by rSmSynt-stimulated cultured splenocytes. The protective effect conferred by vaccination with rSmSynt was demonstrated by 30–37% reduction of worm burden, 38–43% reduction in the number, and 35–37% reduction in the area, of liver granulomas.

Conclusions/Significance

Our report is the first characterization of syntenin in Schistosoma mansoni and our data suggest that this protein is a potential candidate for the development of a multi-antigen vaccine to control schistosomiasis.     

Antischistosomal activity of a calcium channel antagonist on schistosomula and adult Schistosoma mansoni worms

Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+channels.     

Identification of in vivo protein phosphorylation sites in human pathogen Schistosoma japonicum by a phosphoproteomic approach

Schistosome is the causative agent of human schistosomiasis and related animal disease. Reversible protein phosphorylation plays a key role in signaling processing that are vital for a cell and organism. However, it remains to be undercharacterized in schistosomes. In the present study, we characterized in vivo protein phosphorylation events in different developmental stages (schistosomula and adult worms) of Schistosoma japonicum by using microvolume immobilized metal-ion affinity chromatography (IMAC) pipette tips coupled to nanoLC-ESI-MS/MS. In total, 127 distinct phosphorylation sites were identified in 92 proteins in S. japonicum. A comparison of the phosphopeptides identified between the schistosomula and the adult worms revealed 30 phosphoproteins co-detected in both of the two worms. These proteins included several signal molecules and enzymes such as 14-3-3 protein, cysteine string protein, heat shock protein 90, epidermal growth factor receptor pathway substrate 8, proliferation-associated protein 2G4, peptidyl-prolyl isomerase G, phosphofructokinase and thymidylate kinase. Additionally, the phosphorylation sites were examined for phosphorylation specific motif and evolutionarily conservation. The study represents the first attempt to determine in vivo protein phosphorylation in S. japonicum by using a phosphoproteomic approach. The results by providing an inventory of phosphorylated proteins may facilitate to further understand the mechanisms involved in schistosome development and growth, and then may result in the development of novel vaccine candidates and drug targets for schistosomiasis control.     

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.     

Sm10.3, a Member of the Micro-Exon Gene 4 (MEG-4) Family,Induces Erythrocyte Agglutination In Vitro and Partially Protects Vaccinated Mice against Schistosoma mansoni Infection

Background

The parasitic flatworm Schistosoma mansoni is a blood fluke that causes schistosomiasis. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Numerous antigens that are expressed at the interface between the parasite and the mammalian host have been assessed. Among the most promising molecules are the proteins present in the tegument and digestive tract of the parasite.

Methodology/Principal Findings

In this study, we evaluated the potential of Sm10.3, a member of the micro-exon gene 4 (MEG-4) family, for use as part of a recombinant vaccine. We confirmed by real-time PCR that Sm10.3 was expressed at all stages of the parasite life cycle. The localization of Sm10.3 on the surface and lumen of the esophageal and intestinal tract in adult worms and lung-stage schistosomula was confirmed by confocal microscopy. We also show preliminary evidence that rSm10.3 induces erythrocyte agglutination in vitro. Immunization of mice with rSm10.3 induced a mixed Th1/Th2-type response, as IFN-γ, TNF-α, and low levels of IL-5 were detected in the supernatant of cultured splenocytes. The protective effect conferred by vaccination with rSm10.3 was demonstrated by 25.5–32% reduction in the worm burden, 32.9–43.6% reduction in the number of eggs per gram of hepatic tissue, a 23.8% reduction in the number of granulomas, an 11.8% reduction in the area of the granulomas and a 39.8% reduction in granuloma fibrosis.

Conclusions/Significance

Our data suggest that Sm10.3 is a potential candidate for use in developing a multi-antigen vaccine to control schistosomiasis and provide the first evidence for a possible role for Sm10.3 in the blood feeding process.     

Schistosoma mansoni: Interactive effects of irradiation and cryopreservation on parasite maturation and immunization of mice

Mechanically transformed schistosomula of Schistosoma mansoni were irradiated with levels of ⁶⁰Co irradiation between 2.5 and 54 krad, cryopreserved by the two-step addition of ethanediol and rapid cooling technique, and were injected intramuscularly into groups of mice which were perfused 40 days later. The schistosomula were either irradiated and then cryopreserved (IC) or cryopreserved and then irradiated in the frozen state (CI). Development into adult worms was prevented with 4 krad for IC schistosomula, but for CI schistosomula a small number of worms (1.6%) was recovered using 8.8 krad. A dose of 4 krad was sufficient to prevent development of unfrozen controls (I), but for schistosomula irradiated while exposed to ethanediol (EI), a dose of 7 krad was required. Using the different protocols, the peak levels of protection against a challenge infection were achieved with 9 (IC) and 16 krad (CI), compared to 20 krad for unfrozen schistosomula (I) reported previously. The highest level of protection (65%) was achieved with CI schistosomula. Possible interactions between the radioprotective and damaging effects of cryopreservation are discussed.     

The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro

Clegg J. A. and Smithers S. R., 1972. The effect of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. International journal for Parasitology2: 79–98. The sera of rhesus monkeys hyperimmunized by 2–4 exposures to S. mansoni cercariae contain an antibody lethal to schistosomula cultivated in vitro. The antibody (IgG) is dependent on labile factors in fresh monkey serum. It can be absorbed by adult worms cultivated in vitro and it is not the antibody responsible for CHR or COP reactions. A titre of lethal antibody sufficient to kill all schistosomula in vitro is maintained for 2–3 months following challenge: it then falls to a moderate level which may be retained for several years. After inactivation, hyperimmune serum inhibits the growth of cultured schistosomula but does not kill them. Following a small primary infection rhesus serum develops a marked growth-inhibiting property and a low titre of lethal antibody at about 4 months, i.e. the time when resistance to reinfection can first be reliably demonstrated.