Obtenção de anticorpo recombinante (scFv) funcional contra proteínas TcGP35/50 de Trypanosoma cruzi

Orientador: Prof. Dr. Wanderson Duarte da RochaCoorientadora: Profa. Dra. Larissa Magalhães AlvarengaTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências - Bioquímica. Defesa : Curitiba, 06/08/2018Inclui referências: p.87-98Resumo: A doença de Chagas é causada pelo Trypanosoma cruzi e está distribuída em vários países principalmente na América Latina, afetando cerca de 8 milhões de pessoas. Os tratamentos atualmente disponíveis são considerados ineficazes por existirem cepas naturalmente resistentes a esses medicamentos e os critérios de cura não serem efetivos, além disso apresentam diversos efeitos colaterais. Existem diversas formas de transmissão da doença e apesar de vários programas de controle do vetor (inseto barbeiro) essa é ainda a principal forma de contágio. Devido a persistência de várias espécies transmissoras do vetor no ambiente domiciliar e peridomiciliar se faz necessário buscar alternativas de controle que interfiram na proliferação ou na infectividade de formas invasivas presentes no inseto, ou no tratamento de indivíduos com alta parasitemia. Nesse sentido, o objetivo do trabalho é desenvolver anticorpos recombinantes que tenham potencial aplicação na interferência do ciclo de vida do parasito no inseto ou no hospedeiro mamífero. Neste sentido, foi visto que proteínas de superfície como as mucinas (gp35/50) e transialidases (gp82) que participam no processo de adesão e invasão das células possibilitando o estabelecimento da infecção e isso torna candidatos para o desenvolvimento de novas estratégias terapêuticas para o tratamento da doença. Assim, o gene sintético scFv-10D8 previamente construído obtido a partir do mAb-10D8 (anti-gp35/50) foi clonado e expresso em E. coli com proteína de fusão 6xHis (pET22b). Após otimização das condições de indução e enriquecimento de uma fração periplasmática contendo scFv-10D8, foi demonstrado que a proteína recombinante é capaz de reconhecer as mesmas proteínas identificadas pelo mAb-10D8. O ensaio de invasão de células de mamíferos utilizando fração periplasmática ou purificada de scFv10D8 e de um scFv não relacionado foi realizado. Os resultados mostram uma redução específica e dose dependente na taxa de infecção de células expostas a formas tripomastigotas metacíclicos pré-incubados com scFv-10D8. Diante dos resultados apresentados acredita-se que o scFv-10D8 possa ser usado na paratransgênese no inseto vetor evitando disseminação de formas infectivas. Além disso nesse trabalho, as regiões variáveis de IgG das cadeias leves e pesadas de hibridomas que expressam mAb 2B10 (anti-TcGP35/50), e mAb 3F6 (anti-TcGP82) foram sequenciados e montados como scFv. A síntese e produção desses novos scFvs ampliarão as possibilidades de uso dessas moléculas sozinhas ou combinadas com scFv-10D8, associadas ou não a moléculas efetoras (p.ex. peptídeos líticos ao parasito) para controle do parasito no inseto.Abstract: Chagas' disease is caused by Trypanosoma cruzi and is distributed in several countries mainly in Latin America, affecting about 8 million people. The currently available treatments are considered ineffective because there are naturally resistant strains of these drugs and the cure criteria are ineffective. In addition, there are several side effects. There are several forms of transmission of the disease and despite several vector control programs (Triatomine bug) this is still the main form of contagion. Due to the persistence of several vector transmitting species in the home and peridomiciliary environment, it is necessary to search for control alternatives that interfere in the proliferation or infectivity of the invasive forms present in the insect or in the treatment of individuals with high parasitemia. In this sense, the objective of the work is to develop recombinant antibodies that have potential application in the interference of the cycle in the insect or in the treatment of Chagas' disease. In this sense, it was seen that surface proteins such as mucins (gp35/50) and transialidases (gp82) that participate in the process of adhesion and invasion of the cells allowing the establishment of infection and this makes them candidates for the development of new therapeutic strategies for the treatment of the disease. Thus, the previously constructed synthetic gene scFv-10D8 obtained from mAb-10D8 (anti-gp35/50) was cloned and expressed in E. coli with 6xHis fusion protein (pET22b). After optimization of the induction and enrichment conditions of a periplasmic fraction containing scFv- 10D8, it was demonstrated that the recombinant protein is able to recognize the same proteins identified by mAb-10D8. The mammalian cell invasion assay using periplasmic or purified fraction of scFv10D8 and an unrelated scFv was performed. The results show a specific and dose-dependent reduction in the infection rate of cells exposed to metacyclic trypomastigote forms preincubated with scFv-10D8. In view of the presented results, it is believed that the scFv-10D8 can be used in the paratransgênese in the vector insect avoiding dissemination of infective forms. Furthermore, in this work, the light and heavy chain IgG variable regions of hybridomas expressing mAb 2B10 (anti-TcGP35 / 50), and mAb 3F6 (anti-TcGP82) were sequenced and assembled as scFv. The synthesis and production of these new scFvs increase the possibilities of using these molecules alone or combined with scFv-10D8, associated or not with effector molecules (eg lithic peptides to the parasite) to control the parasite in the insect

Obtenção de anticorpo recombinante (scFv) funcional contra proteínas TcGP35/50 de Trypanosoma cruzi

Orientador: Prof. Dr. Wanderson Duarte da RochaCoorientadora: Profa. Dra. Larissa Magalhães AlvarengaTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências - Bioquímica. Defesa : Curitiba, 06/08/2018Inclui referências: p.87-98Resumo: A doença de Chagas é causada pelo Trypanosoma cruzi e está distribuída em vários países principalmente na América Latina, afetando cerca de 8 milhões de pessoas. Os tratamentos atualmente disponíveis são considerados ineficazes por existirem cepas naturalmente resistentes a esses medicamentos e os critérios de cura não serem efetivos, além disso apresentam diversos efeitos colaterais. Existem diversas formas de transmissão da doença e apesar de vários programas de controle do vetor (inseto barbeiro) essa é ainda a principal forma de contágio. Devido a persistência de várias espécies transmissoras do vetor no ambiente domiciliar e peridomiciliar se faz necessário buscar alternativas de controle que interfiram na proliferação ou na infectividade de formas invasivas presentes no inseto, ou no tratamento de indivíduos com alta parasitemia. Nesse sentido, o objetivo do trabalho é desenvolver anticorpos recombinantes que tenham potencial aplicação na interferência do ciclo de vida do parasito no inseto ou no hospedeiro mamífero. Neste sentido, foi visto que proteínas de superfície como as mucinas (gp35/50) e transialidases (gp82) que participam no processo de adesão e invasão das células possibilitando o estabelecimento da infecção e isso torna candidatos para o desenvolvimento de novas estratégias terapêuticas para o tratamento da doença. Assim, o gene sintético scFv-10D8 previamente construído obtido a partir do mAb-10D8 (anti-gp35/50) foi clonado e expresso em E. coli com proteína de fusão 6xHis (pET22b). Após otimização das condições de indução e enriquecimento de uma fração periplasmática contendo scFv-10D8, foi demonstrado que a proteína recombinante é capaz de reconhecer as mesmas proteínas identificadas pelo mAb-10D8. O ensaio de invasão de células de mamíferos utilizando fração periplasmática ou purificada de scFv10D8 e de um scFv não relacionado foi realizado. Os resultados mostram uma redução específica e dose dependente na taxa de infecção de células expostas a formas tripomastigotas metacíclicos pré-incubados com scFv-10D8. Diante dos resultados apresentados acredita-se que o scFv-10D8 possa ser usado na paratransgênese no inseto vetor evitando disseminação de formas infectivas. Além disso nesse trabalho, as regiões variáveis de IgG das cadeias leves e pesadas de hibridomas que expressam mAb 2B10 (anti-TcGP35/50), e mAb 3F6 (anti-TcGP82) foram sequenciados e montados como scFv. A síntese e produção desses novos scFvs ampliarão as possibilidades de uso dessas moléculas sozinhas ou combinadas com scFv-10D8, associadas ou não a moléculas efetoras (p.ex. peptídeos líticos ao parasito) para controle do parasito no inseto.Abstract: Chagas' disease is caused by Trypanosoma cruzi and is distributed in several countries mainly in Latin America, affecting about 8 million people. The currently available treatments are considered ineffective because there are naturally resistant strains of these drugs and the cure criteria are ineffective. In addition, there are several side effects. There are several forms of transmission of the disease and despite several vector control programs (Triatomine bug) this is still the main form of contagion. Due to the persistence of several vector transmitting species in the home and peridomiciliary environment, it is necessary to search for control alternatives that interfere in the proliferation or infectivity of the invasive forms present in the insect or in the treatment of individuals with high parasitemia. In this sense, the objective of the work is to develop recombinant antibodies that have potential application in the interference of the cycle in the insect or in the treatment of Chagas' disease. In this sense, it was seen that surface proteins such as mucins (gp35/50) and transialidases (gp82) that participate in the process of adhesion and invasion of the cells allowing the establishment of infection and this makes them candidates for the development of new therapeutic strategies for the treatment of the disease. Thus, the previously constructed synthetic gene scFv-10D8 obtained from mAb-10D8 (anti-gp35/50) was cloned and expressed in E. coli with 6xHis fusion protein (pET22b). After optimization of the induction and enrichment conditions of a periplasmic fraction containing scFv- 10D8, it was demonstrated that the recombinant protein is able to recognize the same proteins identified by mAb-10D8. The mammalian cell invasion assay using periplasmic or purified fraction of scFv10D8 and an unrelated scFv was performed. The results show a specific and dose-dependent reduction in the infection rate of cells exposed to metacyclic trypomastigote forms preincubated with scFv-10D8. In view of the presented results, it is believed that the scFv-10D8 can be used in the paratransgênese in the vector insect avoiding dissemination of infective forms. Furthermore, in this work, the light and heavy chain IgG variable regions of hybridomas expressing mAb 2B10 (anti-TcGP35 / 50), and mAb 3F6 (anti-TcGP82) were sequenced and assembled as scFv. The synthesis and production of these new scFvs increase the possibilities of using these molecules alone or combined with scFv-10D8, associated or not with effector molecules (eg lithic peptides to the parasite) to control the parasite in the insect

Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion.

Trypanosoma cruzi is a flagellate protozoan pathogen that causes Chagas disease. Currently there is no preventive treatment and the efficiency of the two drugs available is limited to the acute phase. Therefore, there is an unmet need for innovative tools to block transmission in endemic areas. In this study, we engineered a novel recombinant molecule able to adhere to the T. cruzi surface, termed scFv-10D8, that consists of a single-chain variable fragment (scFv) derived from mAb-10D8 that targets gp35/50. The synthetic gene encoding scFv-10D8 was cloned and fused to a 6×His tag and expressed in a prokaryotic expression system. Total periplasmic or 6xHis tag affinity-purified fractions of scFv-10D8 retained the capacity to bind to gp35/50, as shown by Western blot analyses. Pre-incubation of metacyclic trypomastigotes with scFv-10D8 showed a remarkable reduction in cell invasion capacity. Our results suggest that scFv-10D8 can be used in a paratransgenic approach to target parasites in insect vectors, avoiding dissemination of infective forms. Such advances in the development of this functional molecule will surely prompt the improvement of alternative strategies to control Chagas disease by targeting mammalian host stages

Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion

Trypanosoma cruzi is a flagellate protozoan pathogen that causes Chagas disease. Currently there is no preventive treatment and the efficiency of the two drugs available is limited to the acute phase. Therefore, there is an unmet need for innovative tools to block transmission in endemic areas. In this study, we engineered a novel recombinant molecule able to adhere to the T. cruzi surface, termed scFv-10D8, that consists of a single-chain variable fragment (scFv) derived from mAb-10D8 that targets gp35/50. The synthetic gene encoding scFv-10D8 was cloned and fused to a 6×His tag and expressed in a prokaryotic expression system. Total periplasmic or 6xHis tag affinity-purified fractions of scFv-10D8 retained the capacity to bind to gp35/50, as shown by Western blot analyses. Pre-incubation of metacyclic trypomastigotes with scFv-10D8 showed a remarkable reduction in cell invasion capacity. Our results suggest that scFv-10D8 can be used in a paratransgenic approach to target parasites in insect vectors, avoiding dissemination of infective forms. Such advances in the development of this functional molecule will surely prompt the improvement of alternative strategies to control Chagas disease by targeting mammalian host stages