Construção de uma Biblioteca de Anticorpos Anti-ZIKA Vírus
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Data
2019-02-27
Autores
Santos, Francielle Martins
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Bibliotecas conformacionais de anticorpos vem constituindo importantes ferramentas na investigação científica de imunoglobulinas expressas em diferentes situações biológicas incluindo patologias. O conhecimento e elucidação estrutural de anticorpos diferenciais produzidos em patologias distintas pode representar ferramenta molecular fundamental para a intervenção na fisiopatologia da doença; incluindo diagnóstico diferencial, identificação de biomarcadores, terapêutica baseada em imunoglobulinas para bloqueio de patógenos bem como, contribuir para a constituição de painéis de anticorpos capazes de identificar epítopos agregando, também, informações para avanços no desenvolvimento de vacinas. A maior dificuldade atualmente reportada na literatura científica é a obtenção in vitro dos fragmentos Fab (Fragment Antigen Binding) devido à ausência de informações metodológicas das fases experimentais necessárias à construção do Fab, substrato essencial a construção de bibliotecas de anticorpos. Uma abordagem ainda inédita neste contexto é o desenvolvimento de uma metodologia experimental eficiente para a obtenção de bibliotecas de Fab na infecção pelo vírus ZIKA (ZIKV). A infecção pelo vírus ZIKA (ZIKV) vem sendo associada à casos de microcefalia em neonatos de gestantes infectadas e síndrome de Guillain-Barré, a neutralização viral baseada em anticorpos dirigidos contra epítopos específicos é pouco conhecida, a maioria dos estudos se referem a outros Flavivirus. A resposta imunológica humoral é essencial a proteção contra os Flavivirus, por isso é importante conhecer os mecanismos desenvolvidos pelo sistema imunológico do hospedeiro na presença do vírus. Assim, a finalidade deste estudo foi a criação de uma metodologia experimental, com delineamento ainda inédito na literatura, para obtenção do Fab a partir de pacientes com infecção ativa pelo ZIKV e, a obtenção de uma biblioteca de Fab dos anticorpos expressos nestes pacientes. A metodologia utilizada no presente estudo foi adaptada de Andris-Widhopf, et al. (2000) e Barbas et al. (2001). Os resultados obtidos mostraram o desenvolvimento de uma nova metodologia para construção do Fab anti-ZIKV, sugerindo que o detalhamento metodológico é essencial para o sucesso na obtenção da biblioteca, o que inexiste nos artigos anteriores que abordam a construção de bibliotecas conformacionais de anticorpos. Em conclusão a construção do Fab e, de bibliotecas de anticorpos apresentam variabilidades individuais na dependência do patógeno e, das diferentes etapas experimentais do processo.
Conformational libraries of antibodies have become important tools for the scientific investigation of immunoglobulins expressed under different biological situations, including pathological conditions. The knowledge and structural elucidation of differential antibodies produced during distinct pathological conditions may be fundamental for intervention in the pathophysiology of diseases, including differential diagnosis, biomarker identification, immunoglobulin-based therapy for pathogen blocking, and constitution of antibody panels for identifying epitopes for vaccine advancement. Currently, the greatest difficulty reported in the scientific literature is the lack of methodological information for the experimental phases necessary for in vitro construction of Fab (Antigen Binding Fragment), an essential substrate for antibody library construction. In this context, an efficient experimental methodology for obtaining Fab libraries in ZIKA (ZIKV) virus infection is as yet unreported. ZIKV infection has been associated with cases of microcephaly in neonates of infected pregnant women and those with Guillain-Barré syndrome. However, viral neutralization based on antibodies directed against specific epitopes is not well understood, therefore, most studies have been carried out on other Flaviviruses. The humoral immune response is essential for protection against Flaviviruses; hence, it is important to understand the mechanisms of the host immune system against this virus. Thus, the purpose of this study was to develop a novel experimental methodology to obtain Fab from active ZIKV-infected patients and generate a Fab library of the antibodies expressed in these patients. The methodology used in the present study was adapted from those reported by AndrisWidhopf et al. (2000) and Barbas et al. (2001). The results showed the successful development of a new methodology for the construction of anti-ZIKV Fab, suggesting that the methodological details are essential for successfully creating the library. Such details are not presented in previous studies on the construction of conformational libraries of antibodies. In conclusion, the construction of the Fab and antibody libraries presents individual variabilities in the dependence of the pathogen, and of the different experimental stages of the process.
Conformational libraries of antibodies have become important tools for the scientific investigation of immunoglobulins expressed under different biological situations, including pathological conditions. The knowledge and structural elucidation of differential antibodies produced during distinct pathological conditions may be fundamental for intervention in the pathophysiology of diseases, including differential diagnosis, biomarker identification, immunoglobulin-based therapy for pathogen blocking, and constitution of antibody panels for identifying epitopes for vaccine advancement. Currently, the greatest difficulty reported in the scientific literature is the lack of methodological information for the experimental phases necessary for in vitro construction of Fab (Antigen Binding Fragment), an essential substrate for antibody library construction. In this context, an efficient experimental methodology for obtaining Fab libraries in ZIKA (ZIKV) virus infection is as yet unreported. ZIKV infection has been associated with cases of microcephaly in neonates of infected pregnant women and those with Guillain-Barré syndrome. However, viral neutralization based on antibodies directed against specific epitopes is not well understood, therefore, most studies have been carried out on other Flaviviruses. The humoral immune response is essential for protection against Flaviviruses; hence, it is important to understand the mechanisms of the host immune system against this virus. Thus, the purpose of this study was to develop a novel experimental methodology to obtain Fab from active ZIKV-infected patients and generate a Fab library of the antibodies expressed in these patients. The methodology used in the present study was adapted from those reported by AndrisWidhopf et al. (2000) and Barbas et al. (2001). The results showed the successful development of a new methodology for the construction of anti-ZIKV Fab, suggesting that the methodological details are essential for successfully creating the library. Such details are not presented in previous studies on the construction of conformational libraries of antibodies. In conclusion, the construction of the Fab and antibody libraries presents individual variabilities in the dependence of the pathogen, and of the different experimental stages of the process.
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Palavras-chave
Anticorpos, Biblioteca, Fab, ZIKA Vírus, Antibodies, Library, Fab