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Desenvolvimento de um modelo de avalia????o de prot??tipos vacinais em linhagem de mon??cito humana (THP-1)

By Danilo Parmera

Abstract

As culturas de c??lulas v??m sendo utilizadas extensivamente no desenvolvimento e na produ????o de uma variedade de produtos terap??uticos e profil??ticos, tornando-se uma ferramenta indispens??vel para geneticistas, imunologistas, vacinologistas e a ind??stria farmac??utica. A ado????o de sistemas de ensaios celulares in vitrotem sido aplicada como um m??todo alternativo para a substitui????o ou diminui????o do uso de animais nas fases de desenvolvimento, produ????o e testes de vacinas, demonstrando resultados promissores. Da mesma forma, sistemas computacionais podem ampliar a utiliza????o desta ferramenta para etapas do desenvolvimento de vacinas candidatas na fase pr??-cl??nica. O presente trabalho teve como objetivo o desenvolvimento de umprotocolo in vitroutilizando culturas de c??lulas humanas - a linhagem de mon??citos THP-1, para a sele????o de um prot??tipo vacinal - a cepa Pasteur de Mycobacterium bovisBCG expressando o ant??geno Sm14 de Schistossoma mansoni(BCG/sm14). Para isso foram empregadas duas metodologias ??? citometria de fluxo e imunocitoqu??mica, para a identifica????o do perfil da express??o das citocinas IL-10, IL-12 e TNF-??. Foram utilizados como controles a cepa Pasteur do M. bovisBCG e a constru????o da cepa Pasteur do M. bovisBCG contendo o vetor de express??o pAU5 (BCG/pAU5).Ap??s padroniza????o, a multiplicidade de infec????o utilizada para os experimentos foi de 10 bacilos para 1 c??lula THP-1 (10MOI). A express??o daprote??na Sm14 foi detectada em todos os prot??tipos vacinais. Para assegurar queo prot??tipo BCG/sm14 era capaz de diferenciar a linhagem de mon??citos THP-1 em macr??fagos, avaliamos a taxa de crescimento celular e foi poss??vel observarque ap??s a infec????o estas c??lulas apresentavam o ??ndice de prolifera????o diminu??do em 2 logs em rela????o ?? c??lula n??o infectada. O prot??tipo vacinal BCG/sm14n??o mostrou diferen??as quanto a capacidade de infec????o, a taxa de persist??ncia intracelular e a estabilidade da constru????o plasmidial. As duas metodologias empregadas mostraram resultados discrepantes em rela????o ao percentual de citocinas expressas ap??s a infec????o com o prot??tipo vacinal ou os BCG controles, mostrando um maior percentual de c??lulas positivas quando avaliados por citometria de fluxo.Contudo, mesmo com esta diferen??a observamos que o prot??tipo vacinal BCG/sm14 n??o alterou o perfil de express??o de IL-10, IL-12 e TNF-??. O fato do plasm??deo pAU5 ser um vetor de express??o citoplasm??tica, sugere que a prote??na Sm14 n??o foi capaz de estimular a mudan??a de citocinas em mon??citos humanos. Experimentos futuros devem investigar o papel do BCG/sm14em macr??fagos maduros, quanto a indu????o de citocinas pr?? e anti-inflamat??rias, TLR, bem como na indu????o da resposta imune adaptativa, como a apresenta????o antig??nica, na tentativa de melhor entender a resposta imune a esta vacina candidata.Cell cultures has been used extensively in the development of a broad range of therapeutic and prophylactic products, and are an important tool for geneticists, immunologists, vaccinologists and the pharmaceuticsindustry. In vitrocell assay has been applied as an alternative method to replace ordiminish the use of animal model in the developmental, production in vaccine test phases, with promising results. Otherwise, computational methods should amplify theuse of cell cultures tools to test candidate vaccines. The mean goal of this work was to develop an in vitro protocol using human cell line ??? monocytic cell THP-1, to select a vaccine prototype ??? strain Pasteur Mycobacterium bovisBCG expressing Schistosoma mansoniSm14-antigen (BCG/sm14). Two methodologies were employed ??? a flow cytometry and immunocytochemistry, to quantify the expression of IL-10, IL-12 e TNF-??. Pasteur M. bovisBCG and the strain Pasteur do M. bovisBCG containing the expression vector pAU5 (BCG/pAU5) were used as controls. Multiplicityof infection (MOI) was determined and showed a better 10 bacilli to 1 cells ratios, regarding the expression of intracellular cytokines. Sm14 protein expressionwas detected in all vaccine prototype before use. To assure that BCG/sm14was able to differentiate monocytic THP1 cell line in mature macrophage, we evaluated the proliferative ration after BCGs infection and all strain showed the ability toinduce monocytic THP1 cells maturation, diminishing in 2.0 logs the cell proliferation. No differences were seen in the uptake, intracellular persistence and plasmidial stability. Interestingly, we observe discrepant results regarding the amount of positivecells expressing cytokines detected by the two methods used, independent of which BCG was tested. The overall results obtained by the cytometric method was high than immunocytochemistry. However, beside these ambiguous results, no alteration in the IL-10, IL-12 and TNF-alfa profile was observed whenBCG/sm14was compared with BCG. These results point to the plamidial BCG construction pAU5 and its intracellular expression, suggesting no modification in the cytokine profile in human monocytic cell lines. Further experiments should be addressedto identify the role of BCG/sm14 in modulate mature macrophage and, the induction ofadaptive immune response, as antigen presentation, pro- and anti-inflammatory cytokines, TLR expression and activation, to better understating the vaccine prototype immune response

Topics: THP-1, Vacina BCG, Sm-14, Vacina, Prot??tipo, Citocinas, THP-1, BCG Vaccine, Sm-14, Vaccine, Prototype, Cytokines, Vacina BCG, Citocinas
Publisher: Instituto de Tecnologia em Imunobiol??gicos
Year: 2007
OAI identifier: oai:agregador.ibict.br.RI_FIOCRUZ:oai:localhost:icict/5808
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