Molecular cloning of components of protective antigenic preparations has suggested that related parasite fatty acid-binding proteins could form the basis of the protective immune crossreactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. Molecular models of the two parasite proteins showed that both molecules adopt the same basic three-dimensional structure, consisting of a barrel-shaped molecule formed by 10 antiparallel (8-pleated strands joined by short loops, and revealed the likely presence of crossreactive, discontinuous epitopes principally derived from amino acids in the C-terminal portions of the molecules. A recombinant form of the S. mansoni antigen, rSml4, protected outbred Swiss mice by up to 67% against challenge with S. mansoni cercariae in the absence of adjuvant and without provoking any observable autoimmune response. The same antigen also provided complete protection against challenge with F. hepatica metacercariae in the same animal model. The results suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni, of veterinary and human importance, respectively. Schistosomiasis, caused principally by Schistosoma mansoni, S. haematobium, and S. japonicum, afflicts some 200 million individuals in tropical regions of the world. Fascioliasis caused by Fasciola hepatica is an economically important disease of cattle and sheep in Europe, the Americas, Australia, and New Zealand. There are no vaccines against Schistosoma or Fasciola species; however, there is evidence for protective immune crossreactivity between S. mansoni and F. hepatica. Hillyer and coworkers (1-3) have isolated a low molecular weight F. hepatica fraction that protects against both S. mansoni and F. hepatica infections. A component of this fraction is an antigen with homology to mammalian fatty acid-binding proteins that is termed Fhl5 (4). A similar antigen, Sm14, was cloned from S. mansoni following studies of a protective saline extract of adult worms, SE (5). These results suggested that the pair of similar parasite proteins could mediate immune crossreaction and represent the basis of a subunit vaccine effective against both species. We have investigated the molecular relationship of Fhl5, Sml4, and mammalian fatty acid-binding proteins (FABPs) (6) (9), and rat intestine (10) were obtained directly from the Brookhaven Protein Data Bank (accession codes 2HMB, 1ALB, and 2IFB, respectively). Sequences of known crystal structure were aligned by leastsquares superposition of the molecules using C' coordinates alone, and the remaining sequences were subsequently incorporated into the alignment by the method of Barton and Sternberg (11) as implemented in the AMPS package.For the construction of the Sm14 model, the backbone of the 10 (3-strands and three a-helices was based on that of 1ALB. (3-Turn types were determined on the basis of the position of glycine and/or asparagine and aspartic residues within ...

The Sm14 antigen of Schistosoma mansoni was cloned and expressed in Mycobacterium bovis BCG as a fusion with the Mycobacterium fortuitum ␤-lactamase protein under the control of its promoter, pBlaF*; the protein was localized in the bacterial cell wall. The rBCG-Sm14 strain was shown to be relatively stable in cultured murine and bovine monocytes in terms of infectivity, bacterial persistence, and plasmid stability. The immunization of mice with rBCG-Sm14 showed no induction of anti-Sm14 antibodies; however, splenocytes of immunized mice released increased levels of gamma interferon upon stimulation with recombinant Sm14 (rSm14), indicating an induction of a Th1-predominant cellular response against Sm14. Mice immunized with one or two doses of rBCG-Sm14 and challenged with live S. mansoni cercaria showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of rSm14 purified from Escherichia coli. The data presented here further enhance the status of Sm14 as a promising candidate antigen for the control of schistosomiasis and indicate that a one-dose regimen of rBCG-Sm14 could be considered a convenient means to overcome many of the practical problems associated with the successful implementation of a multiple-dose vaccine schedule in developing countries.

The Schistosoma mansoni Sm14 antigen belongs to the fatty acid-binding protein family and is considered a vaccine candidate against at least two parasite worms, Fasciola hepatica and S. mansoni. Here the genomic sequence and the polymorphism of Sm14 have been characterized for the first time. We found that the conserved methionine at position 20 is polymorphic, being exchangeable with threonine (M20T). To evaluate the function of the amino acid residue at this position, we have also constructed the mutant Sm14-A20 besides the two native isoforms (Sm14-M20 and Sm14-T20). The three purified recombinant His 6 -tagged Sm14 proteins (rSm14-M20, rSm14-T20, and rSm14-A20) present a predominant ␤-barrel structure as shown by CD spectroscopy. Thermal and urea unfolding studies evidenced a higher structural stability of rSm14-M20 over the other forms (rSm14-M20>rSm14-T20>rSm14-A20). All of the Sm14 proteins were able to bind 11-(dansylamino)undecanoic acid (DAUDA) without substantial difference in the binding affinity. However, rSm14-M20 exhibited a higher affinity for natural fatty acids than the rSm14-T20 and rSm14-A20 proteins as judged by competitive experiments against DAUDA (rSm14-M20>rSm14-T20> rSm14-A20). The rSm14-M20 or rSm14-T20 isoforms but not the rSm14-A20 mutant was able to induce significant protection against S. mansoni cercariae challenge in immunized mice. The level of protection efficacy correlates with the extent of structure stability of the recombinant Sm14 isoforms and mutant.

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Biomphalaria glabrata is the main intermediate host involved in schistosomiasis in Brazil. Studies have shown that physiological stress conditions, such as infection with Schistosoma mansoni, starvation, aestivation and exposure to molluscicides, can alter its carbohydrate metabolism (Schwartz & Carter 1982, Becker 1983, Bezerra et al. 1999, Alcanfor 2001, Mello-Silva et al. 2006a). These changes can alter the glycogenesis, gluconeogenesis and glycolysis in the snail.One of most promising Brazilian molluscicides is the crude extract of Euphorbia splendens (Sin. Euphorbia milii), which under laboratory and field conditions meets the recommendations of the WHO for use as a natural molluscicide (Vasconcellos & Amorim 2003). Mello-Silva et al. (2006a, 2007 studied the influence of sublethal doses of the latex of this plant on the carbohydrate and protein metabolism and reproductive biology of B. glabrata and found strong metabolic changes leading to a reduction in its population and consequently in the transmission of the parasite. In spite of this potential, there are no studies of the influence of this product on the physiology of Biomphalaria infected with S. mansoni.The present paper examines the action of the latex of E. splendens var. hislopii on the glucose content of the haemolymph and on the carbohydrate (glycogen) deposits of B. glabrata (BH strain) infected with S. mansoni (BH strain). MATERIALS AND METHODSObtaining the latex of E. splendens var. hislopiiSamples of E. splendens var. hislopii latex were collected in the autumn from plants cultivated in plots near the Biology Department, Fiocruz, in Rio de Janeiro, Brazil. The latex was collected as described by Vasconcellos and Amorim (2003) on the same day the tests were conducted.Lethal dose experiment -Using the recently collected latex, an aqueous stock solution at a concentration of 100 mg/L was prepared and, from this, solutions of different concentrations were prepared for use in the bioassays. The lethal (LC 90 ) and sublethal (LC 50 ) concentrations were determined according to Vasconcellos and Amorim (2003), as recommended by the World Health Organization (1983) and Mott (1987). The LC 50 and LC 90 values were, respectively, 1.0 mg/L and 2.3 mg/L (Mello-Silva et al. 2006a).Balloon flasks (1000 mL) were used and the latex solution was divided into two 500 mL glass beakers. The groups of B. glabrata (BH lineage), infected and uninfected, respectively, were placed in LC 50 solutions and exposed for 24 h (Vasconcellos & Amorim 2003) at 21ºC. Two glass beakers received 500 mL of distilled water without latex as a control group. None of the snails was fed during this period.After the latex exposure period, the snails were removed from the flasks and rinsed in distilled water to remove the residues. The number of dead specimens was noted. (1990). The snails were grouped according to their infection stage (1 day and 1, 2, 3 and 4 weeks post exposure). In each period analysed, 100 infected and 20 uninfected snails (control) were used. Sixty infected sn...

Fasciola hepatica is the causative agent of fasciolosis in many areas in America, Europe, Africa, Asia and Australia. There is an urgent need for improved methods to control the parasite's transmission. We describe the use of an experimental vaccine based on a recombinant antigen cloned from another parasite, Schistosoma mansoni (Sm14), that induces high levels of cross protection in mice against both S. mansoni and F. hepatica. Sheep and mice vaccinated with Sm14 were significantly protected against challenge infection with metacercariae of Fasciola hepatica and were completely free of the histopathological hepatic damage related to liver fluke infection. The vaccine will provide a valuable new tool to aid in transmission control of this economically important disease.

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Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system