Construction and evaluation of rLTB/Sm14: a recombinant chimera candidate vaccine against schistosomiasis and fascioliasis

A esquistossomose, causada por Schistosoma mansoni, ?? a segunda doen??a parasit??ria mais prevalente no mundo causando doen??a cr??nica em milh??es de pessoas em pa??ses em desenvolvimento. Semelhantemente, a fasciolose, causada pelo tremat??deo Fasciola hepatica, representa um reconhecido problema agr??cola respons??vel por perdas econ??micas assim como causa um significante n??mero de infec????es em humanos por todo mundo. Embora a quimioterapia para ambos os parasitas esteja dispon??vel, ela tem apresentado limita????es, incluindo altas taxas de reinfec????o em ??reas end??micas. Vacinas representam a alternativa mais atraente para inverter este cen??rio. Assim, a Organiza????o Mundial da Sa??de selecionou a prote??na ligadora de ??cidos graxos 14kDa de S. mansoni (Sm14) como uma de duas candidatas ?? vacina anti-esquistossomose priorit??rias para triagem cl??nica em humanos. Al??m disso, a Sm14 ?? a ??nica prote??na candidata a vacina que induz imunidade protetora significativa contra ambos os helmintos acima mencionados. O objetivo deste estudo foi desenvolver e avaliar em camundongos uma vacina de subunidade contendo a Sm14 fusionada com a subunidade B da enterotoxina termol??bil de Escherichia coli (LTB), a qual ?? um potente imunoadjuvante; al??m disso, foi descrever a produ????o e caracteriza????o de anticorpos monoclonais (MAbs) contra a Sm14 para usa-los como uma ferramenta para detectar e caracterizar a Sm14 e para o desenvolvimento de futuro teste diagn??stico. Os genes ltb e sm14 foram amplificados por PCR a partir do DNA de E. coli e do plasm??deo pAESm14, respectivamente, e fusionados por PCR. A quimera recombinante foi expressa em E. coli e purificada por cromatografia de afinidade em coluna com n??quel. Grupos de camundongos su????os n??o sing??nicos foram imunizados por via subcut??nea com tr??s doses de rLTB/Sm14, rLTB/Sm14 mais hidr??xido de alum??nio (Al(OH)3), rSm14 plus Al(OH)3, rLTB ou Al(OH)3. Os n??veis de prote????o foram determinados atrav??s do n??mero de parasitas recuperados ap??s desafio com cerc??rias de S. mansoni. O pool de soros dos camundongos imunizados foi avaliado por ELISA e Western blot. Os resultados mostraram que a prote??na quimera foi capaz de estimular a produ????o de anticorpos espec??ficos para Sm14 e LTB, contudo o uso de LTB como adjuvante para imuniza????o de rSm14 falhou em aumentar a prote????o contra o desafio. Al??m disso, sete MAbs foram obtidos ap??s imuniza????o de um camundongo BALB/c com rLTB/Sm14. A isotipagem dos MAbs revelou que cinco foram do isotipo IgG1, um pertencente ao isotipo IgG2b e outro ao isotipo IgM. Estes MAbs ser??o ??teis para monitorar a express??o da prote??na em sistemas recombinantes, a estabilidade prot??ica e pode ter potencial para desenvolver teste diagn??stico.Schistosomiasis, caused by Schistosoma mansoni, is the second most prevalent parasitic disease worldwide causing chronic disease in millions of people in developing countries. Similarly, fascioliasis, caused by the trematode Fasciola hepatica, represents a recognized unsolved agricultural problem responsible for economic losses as well as causing a significant number of human infections worldwide. Although chemotherapy for both parasites has been available, this approach has limitations, including high reinfection rates in endemic areas. Vaccines represent the most attractive long-term alternative to invert this scenario. Accordingly, the World Health Organization selected S. mansoni fatty acid-binding protein 14kDa (Sm14) as one out of two anti-schistosome vaccine priority candidates for human clinical trials. Furthermore, Sm14 is the only vaccine candidate to have been shown to afford significant immune protection against both of the above-mentioned helminthes. The objective of this study was to develop and evaluate in mice a recombinant subunit vaccine containing the Sm14 fused to the B subunit of the heat-labile enterotoxin 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 for development of future diagnostic test. The ltb and sm14 gene were obtained by PCR amplification from E. coli DNA and plasmid pAESm14, respectively, and fused by PCR. The recombinant chimera was expressed in E. coli and purified by nickel affinity chromatography. Groups of outbreed Swiss mice were immunized with three footpad doses of either rLTB/Sm14, rLTB/Sm14 plus aluminum hydroxide (Al(OH)3), rSm14 plus Al(OH)3, rLTB or Al(OH)3. Levels of protection were determined by number of worms recovered after S. mansoni cercarial challenge. The pooled sera of immunized mice were evaluated by ELISA and Western blot. The results showed that the chimera protein was able to elicit specific antibody production to rSm14 and rLTB, however the use of LTB as adjuvant to rSm14 immunization failed to enhance protection against challenge. Furthermore, seven MAbs were obtained after immunization of BALB/c mice with rLTB/Sm14. MAbs isotyping revealed that five were of the isotype IgG1, one belonged to the IgG2b isotype and another to the IgM isotype. These MAbs will be usefull for monitoring protein expression in recombinant systems, protein stability and may have potential for developing diagnostic test