Immunogenicity, antibody responses and vaccine efficacy of recombinant annexin B30 against Schistosoma mansoni.

AIMS: Schistosomes infect approximately 250 million people worldwide. To date, there is no effective vaccine available for the prevention of schistosome infection in endemic regions. There remains a need to develop means to confer long-term protection of individuals against reinfection. In this study, an annexin, namely annexin B30, which is highly expressed in the tegument of Schistosoma mansoni was selected to evaluate its immunogenicity and protective efficacy in a mouse model. METHODS AND RESULTS: Bioinformatics analysis showed that there were three potential linear B-cell epitopes and four conformational B-cell epitopes predicted from annexin B30, respectively. Full-length annexin B30 was cloned and expressed in Escherichia coli BL21(DE3). In the presence of adjuvants, the soluble recombinant protein was evaluated for its protective efficacy in two independent vaccine trials. Immunization of CBA mice with recombinant annexin B30 formulated either in alum only or alum/CpG induced a mixed Th1/Th2 cytokine profile but no significant protection against schistosome infection was detected. CONCLUSION: Recombinant annexin B30 did not confer significant protection against the parasite. The molecule may not be suitable for vaccine development. However, it could be an ideal biomarker recommended for immunodiagnostics development.

Evaluation of Lignans from Piper cubeba against Schistosoma mansoni Adult Worms: A Combined Experimental and Theoretical Study.

Six dibenzylbutyrolactonic lignans ((-)-hinokinin (1), (-)-cubebin (2), (-)-yatein (3), (-)-5-methoxyyatein (4), dihydrocubebin (5) and dihydroclusin (6)) were isolated from Piper cubeba seed extract and evaluated against Schistosoma mansoni. All lignans, except 5, were able to separate the adult worm pairs and reduce the egg numbers during 24 h of incubation. Lignans 1, 3 and 4 (containing a lactone ring) were the most efficient concerning antiparasitary activity. Comparing structures 3 and 4, the presence of the methoxy group at position 5 appears to be important for this activity. Considering 1 and 3, it is possible to see that the substitution pattern change (methylenedioxy or methoxy groups) in positions 3' and 4' alter the biological response, with 1 being the second most active compound. Computational calculations suggest that the activity of compound 4 can be correlated with the largest lipophilicity value.

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.

Cloning of Schistosoma mansoni Seven in Absentia (SmSINA)(+) homologue cDNA, a gene involved in ubiquitination of SmRXR1 and SmRXR2.

Drosophila (SINA) and human Seven in Absentia (SIAH-1 and SIAH-2) have been implicated in ubiquitin-mediated proteolysis of different target proteins. Using the Schistosoma mansoni nuclear receptor SmRXR2 as bait in a yeast two-hybrid system, we identified a DNA fragment that encodes part of the schistosome homologue of the Seven in Absentia protein (SmSINA). Screening of S. mansoni cDNA expression library resulted in the isolation of a cDNA containing the full-length coding region of SmSINA. SmSINA contains the characteristic structural features of other SINA proteins including a conserved N-terminal RING finger domain and a cysteine-rich C-terminus. We demonstrate that SmSINA associates with SmRXR2 and SmRXR1 both in vivo and in vitro, and define the binding domains in SmRXR2 and SmRXR1 that mediate their interaction. Schistosome SINA co-localizes with SmRXR2 and SmRXR1 in vitelline cells. In addition, we show that SmSINA stimulates the ubiquination of both SmRXR2 and SmRXR1 in vitro. Our findings suggest that SmSINA regulates ubiquitination and ubiquitin-induced degradation of schistosome nuclear receptors (RXR1 and RXR2) via the ubiquitin-proteasome pathway.

Cloning and characterization of a novel form of tyrosine hydroxylase from the human parasite, Schistosoma mansoni.

Catecholamines such as dopamine and noradrenaline play important roles as neuromuscular transmitters and modulators in all parasitic helminths, including the human parasite, Schistosoma mansoni. We have cloned a novel S. mansoni tyrosine hydroxylase (SmTH) cDNA that shows high homology to mammalian tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Two subsets of SmTH transcripts were identified, one of which carries the S. mansoni spliced-leader (SL) sequence at its 5' end, whereas the other does not appear to be trans-spliced to the S. mansoni SL. The two types of SmTH transcripts encode the same protein of 465 amino acids and a predicted size of 54 kDa. Expression of SmTH as an N-terminal histidine fusion protein in Escherichia coli produced an active enzyme that was purified approximately 52-fold to apparent homogeneity and had a final specific activity of 0.78 micromol/min/mg. The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and the same sensitivity to inhibition by high concentrations of the substrate, tyrosine, as the mammalian enzyme. Purified SmTH also showed characteristic inhibition by catecholamine products, although the sensitivity to product inhibition was lower than that of the mammalian enzyme. This evidence indicates that SmTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian host's enzyme but may differ in its properties of regulation. This first demonstration of tyrosine hydroxylase in a parasitic helminth further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.

A selenium-containing phospholipid-hydroperoxide glutathione peroxidase in Schistosoma mansoni.

The 100000Xg supernatant parasite platyhelminth Schistosoma mansoni exhibits a glutathione peroxidase activity with the substrate phosphatidylcholine hydroperoxide. Purification yielded a protein of 20 kDa molecular mass both on gel filtration column chromatography and SDS/PAGE, thus suggesting that S. mansoni expresses a protein similar to the mammalian selenoenzynic phospholipid-hydroperoxide glutathione peroxidase. Kinetic analysis and substrate specificity corroborated this assumption, the second-order rate constants for the oxidation of the ground-state enzyme (k+1) being higher with phosphatidylcholine hydroperoxide than with other peroxide substrates, such as cumene liydroperoxide or H2O2, and quantitatively similar to those of mammalian phospholipid-hydroperoxide glutathione peroxidase. Partial sequencing of the protein and selenium measurement by neutron activation analysis established that the purified peroxidase corresponded to the product of the S. mansoni gene previously reported and supposed to encode a selenium-containing glutathione peroxidase [Roche, C., Williams, D. L., Khalife, J., LePresle, T., Capron, A. & Pierce, R. J. (1994) Cloning and characterization of gene encoding Schistosoma mansoni glutathione peroxidase, Gene 138, 149 - 152]. S. mansoni thus contains a scienoperoxidase sharing molecular mass, catalytic efficiency and substrate specificity with phospholipid-hydroperoxide glutathione peroxidase, dismantling the concept that those enzymes are unique to vertebrate organisms.