Construction and evaluation of rLTB/Sm14: arecombinant chimera candidate vaccine against schistosomiasis and fascioliasis
Schistosomiasis, caused by Schistosoma mansoni, is the second most prevalent parasiticdisease worldwide causing chronic disease in millions of people in developing countries.Similarly, fascioliasis, caused by the trematode Fasciola hepatica, represents a recognizedunsolved agricultural problem responsible for economic losses as well as causing asignificant number of human infections worldwide. Although chemotherapy for bothparasites has been available, this approach has limitations, including high reinfectionrates in endemic areas. Vaccines represent the most attractive long-term alternative toinvert this scenario. Accordingly, the World Health Organization selected S. mansoni fattyacid-binding protein 14kDa (Sm14) as one out of two anti-schistosome vaccine prioritycandidates for human clinical trials. Furthermore, Sm14 is the only vaccine candidate tohave been shown to afford significant immune protection against both of the above-mentionedhelminthes. The objective of this study was to develop and evaluate in mice arecombinant subunit vaccine containing the Sm14 fused to the B subunit of the heat-labileenterotoxin of Escherichia coli (LTB), which is a potent immunoadjuvant; furthermore,was describe the production and characterization of monoclonal antibodies (MAbs)against the Sm14 for use as a tool to detect and characterize Sm14 and fordevelopment of future diagnostic test. The ltb and sm14 gene were obtained by PCRamplification from E. coli DNA and plasmid pAESm14, respectively, and fused by PCR.The recombinant chimera was expressed in E. coli and purified by nickel affinitychromatography. Groups of outbreed Swiss mice were immunized with three footpaddoses of either rLTB/Sm14, rLTB/Sm14 plus aluminum hydroxide (Al(OH)3), rSm14 plusAl(OH)3, rLTB or Al(OH)3. Levels of protection were determined by number of wormsrecovered after S. mansoni cercarial challenge. The pooled sera of immunized mice wereevaluated by ELISA and Western blot. The results showed that the chimera protein wasable to elicit specific antibody production to rSm14 and rLTB, however the use of LTB asadjuvant to rSm14 immunization failed to enhance protection against challenge.Furthermore, seven MAbs were obtained after immunization of BALB/c mice withrLTB/Sm14. MAbs isotyping revealed that five were of the isotype IgG1, one belonged tothe IgG2b isotype and another to the IgM isotype. These MAbs will be usefull formonitoring protein expression in recombinant systems, protein stability and may havepotential for developing diagnostic test.
Advisor:Odir Antônio Dellagostin; José Antonio Guimarães Aleixo; Fabio Pereira Leivas Leite; Fabrício Rochedo Conceição
School:Universidade Federal de Pelotas
Source Type:Master's Thesis
Keywords:Schistosoma mansoni Biotechnology Schistossomiasis Vaccine Monoclonal antibodies
Date of Publication:10/30/2006