Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue

Detalhes bibliográficos
Ano de defesa: 2020
Autor(a) principal: Lopes, Patrícia da Silva
Orientador(a): Não Informado pela instituição
Banca de defesa: Não Informado pela instituição
Tipo de documento: Tese
Tipo de acesso: Acesso aberto
Idioma: por
Instituição de defesa: Universidade Federal de Uberlândia
Brasil
Programa de Pós-graduação em Ciências da Saúde
Programa de Pós-Graduação: Não Informado pela instituição
Departamento: Não Informado pela instituição
País: Não Informado pela instituição
Palavras-chave em Português:
Link de acesso: https://repositorio.ufu.br/handle/123456789/31127
http://doi.org/10.14393/ufu.te.2020.3021
Resumo: Arboviruses caused by the Dengue virus (DENV) and Zika (ZIKV), are currently considered diseases that represent a potential challenge to public health, resulting in intense and limiting clinical manifestations, which can culminate in microcephaly (ZIKV) and death. There are still no vaccines in the public network or any other preventive drug, the diagnosis is inaccurate due to the cross reactions between the antibodies of DENV, ZIKV and other flaviviruses. Given this context, we aim to develop new molecules based on small peptides (phage) that bind to the ZIKV and DENV-2 virus using the Phage-Display methodology. Thus, the intention of the present study was to select, characterize and validate ZIKV and DENV-2 ligands, assessing their ability to inhibit and also differentiate ZIKV and DENV. The ELISA immunoassay, ZIKV and DENV-2 entry inhibition assay in vitro by flow cytometry using Vero cells and 4G2 antibody and silica analysis were used for the validation of the selected phages. ELISA was also performed with a phage that did not show significant viral inhibition and another that was effective for inhibition using samples from patients infected with DENV and ZIKV. Phage D4, selected for ZIKV, was able to inhibit the entry of ZIKV and DENV-2. F12 phage, also selected for ZIKV, was not able to inhibit ZIKV entry, however it was able to discriminate DENV patients from ZIKV patients and healthy individuals (p <0.0001) with high accuracy, presenting 94.9% specificity and 81.2% sensitivity for DENV infection. The G8 phage selected for DENV-2 was able to inhibit the entry of DENV-2 and also discriminated against DENV from ZIKV and healthy patients in a preliminary test that will be optimized with a larger number of samples. The silica study showed that the possible interaction sites of the peptides expressed by the D4 phage, which inhibit the entry of ZIKV and DENV, were located in domain III of the envelope protein (EDIII) or very close to the fusion loop. Both EDIII and the fusion loop, which is located in EDII, are important target epitopes for antibodies already described in the literature with wide neutralizing action for flavivirus. In the study of phages F12 and G8, which differentiated DENV patients from ZIKV and healthy individuals, we observed their interactions with amino acid residues from the ZIKV and DENV-2 envelope protein, which comprise the kl loop, which is discussed in comparative studies of Zika and Dengue viruses. Through these studies we will seek the integration of important methodologies, covering low cost, speed and high efficiency, in order to fill possible gaps and demands that we still find in the diagnosis of DENV and ZIKV in different stages of the infection, as well as to contribute to future complementary studies, in order to produce possible therapeutic and preventive interventions. In general, this study demonstrated that Phage-Display technology is a strong tool for the development of molecules with antiviral and diagnostic potential. With this, the next step will be to synthesize the peptides expressed in the phages, carrying out tests that can contribute to the development of vaccines for ZIKV, DENV or even for other infectious agents from synthetic molecules.
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spelling Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e DengueDevelopment of molecules with teranostic potential for zika and dengue virusesPhage-DisplayPhage-Displayvírus DengueDengue virusvírus ZikaZika virusInibiçãoInhibitionDiagnósticoDiagnosticCNPQ::CIENCIAS DA SAUDEVírus da ZikaDengueArboviruses caused by the Dengue virus (DENV) and Zika (ZIKV), are currently considered diseases that represent a potential challenge to public health, resulting in intense and limiting clinical manifestations, which can culminate in microcephaly (ZIKV) and death. There are still no vaccines in the public network or any other preventive drug, the diagnosis is inaccurate due to the cross reactions between the antibodies of DENV, ZIKV and other flaviviruses. Given this context, we aim to develop new molecules based on small peptides (phage) that bind to the ZIKV and DENV-2 virus using the Phage-Display methodology. Thus, the intention of the present study was to select, characterize and validate ZIKV and DENV-2 ligands, assessing their ability to inhibit and also differentiate ZIKV and DENV. The ELISA immunoassay, ZIKV and DENV-2 entry inhibition assay in vitro by flow cytometry using Vero cells and 4G2 antibody and silica analysis were used for the validation of the selected phages. ELISA was also performed with a phage that did not show significant viral inhibition and another that was effective for inhibition using samples from patients infected with DENV and ZIKV. Phage D4, selected for ZIKV, was able to inhibit the entry of ZIKV and DENV-2. F12 phage, also selected for ZIKV, was not able to inhibit ZIKV entry, however it was able to discriminate DENV patients from ZIKV patients and healthy individuals (p <0.0001) with high accuracy, presenting 94.9% specificity and 81.2% sensitivity for DENV infection. The G8 phage selected for DENV-2 was able to inhibit the entry of DENV-2 and also discriminated against DENV from ZIKV and healthy patients in a preliminary test that will be optimized with a larger number of samples. The silica study showed that the possible interaction sites of the peptides expressed by the D4 phage, which inhibit the entry of ZIKV and DENV, were located in domain III of the envelope protein (EDIII) or very close to the fusion loop. Both EDIII and the fusion loop, which is located in EDII, are important target epitopes for antibodies already described in the literature with wide neutralizing action for flavivirus. In the study of phages F12 and G8, which differentiated DENV patients from ZIKV and healthy individuals, we observed their interactions with amino acid residues from the ZIKV and DENV-2 envelope protein, which comprise the kl loop, which is discussed in comparative studies of Zika and Dengue viruses. Through these studies we will seek the integration of important methodologies, covering low cost, speed and high efficiency, in order to fill possible gaps and demands that we still find in the diagnosis of DENV and ZIKV in different stages of the infection, as well as to contribute to future complementary studies, in order to produce possible therapeutic and preventive interventions. In general, this study demonstrated that Phage-Display technology is a strong tool for the development of molecules with antiviral and diagnostic potential. With this, the next step will be to synthesize the peptides expressed in the phages, carrying out tests that can contribute to the development of vaccines for ZIKV, DENV or even for other infectious agents from synthetic molecules.Tese (Doutorado)As arboviroses causadas pelo vírus Dengue (DENV) e Zika (ZIKV), são atualmente consideradas doenças que representam um potencial desafio para a saúde pública, resultando em intensas e limitantes manifestações clínicas, podendo culminar em microcefalia (ZIKV) e óbito. Ainda não existem vacinas na rede pública ou qualquer outra droga preventiva, o diagnóstico é impreciso devido as reações cruzadas entre os anticorpos de DENV, ZIKV e de outros flavivírus. Diante desse contexto, objetivamos desenvolver e também testar novas moléculas baseadas em pequenos peptídeos (fagos) ligantes ao vírus ZIKV e DENV-2 utilizando a metodologia Phage-Dispay. Assim, o objetivo do presente estudo foi selecionar, caracterizar e validar ligantes de ZIKV e DENV-2, bem como testar fagos miméticos a um inibidor de fosfolipase A2 avaliando a capacidade dessas moléculas em inibir e também diferenciar ZIKV e DENV. O imunoensaio ELISA, ensaio de inibição da entrada de ZIKV e DENV-2 in vitro por citometria de fluxo utilizando células Vero e anticorpo 4G2 e análises in sílico foram utilizados para a validação dos fagos selecionados. Também foi realizado ELISA utilizando amostras de pacientes infectados com DENV e ZIKV. Notavelmente, os fagos testados foram capaz de diferenciar DENV de ZIKV e de indivíduos saudáveis. A acurácia no diagnóstico foi determinada pela curva ROC. O fago D4, selecionado para ZIKV, foi capaz de inibir a entrada de ZIKV e DENV-2. O fago F12, também selecionado para ZIKV, não foi capaz de inibir a entrada de ZIKV, entretanto foi capaz de discriminar pacientes de DENV de pacientes de ZIKV e indivíduos saudáveis (p<0.0001) com alta acurácia, apresentando 94,9% de especificidade e 81,2% de sensibilidade para infecção por DENV. O fago G8 selecionado para DENV-2, foi capaz de inibir a entrada de DENV-2 e também discriminou DENV de ZIKV e pacientes saudáveis em um teste preliminar que será otimizado com maior número de amostras. O estudo in sílico mostrou os possíveis locais de interação dos peptídeos expressos pelo fago D4, que inibe a entrada de ZIKV e DENV, apresentando interação com o domínio III da proteína de envelope (EDIII) ou muito próximo ao loop de fusão. Tanto EDIII quanto o loop de fusão, que está localizado em EDII são importantes epítopos alvos para anticorpos já descritos na literatura de ampla ação neutralizates para flavivírus. Já no estudo dos fagos F12 e G8, que diferenciaram pacientes de DENV de ZIKV e indivíduos saudáveis observamos suas interações com resíduos de aminoácidos da proteína de envelope de ZIKV e DENV-2, que compreendem o loop kl, que é discutidos em estudos de comparações dos vírus Zika e Dengue. Através desses estudos buscaremos a integração de importantes metodologias, abrangendo baixo custo, rapidez e alta eficiência, a fim de preencher possíveis lacunas e demandas que ainda encontramos no diagnóstico de DENV e ZIKV em diferentes fases da infecção, bem como contribuir para futuros estudos complementares, a fim de produzir possíveis intervenções terapêuticas e preventivas. Em geral, este estudo demonstrou que a tecnologia Phage-Display é uma ferramenta forte para o desenvolvimento de moléculas com potencial antiviral e diagnóstico. Com isso, o próximo passo será sintetizar os peptídeos expressos nos fagos, realizando testes que podem contribuir para o desenvolvimento de vacinas para ZIKV, DENV ou mesmo para outros agentes infeciosos a partir de moléculas sintéticas.2022-05-29Universidade Federal de UberlândiaBrasilPrograma de Pós-graduação em Ciências da SaúdeSantos, Paula Souzahttp://lattes.cnpq.br/5065438852500935Goulart Filho, Luiz Ricardohttp://lattes.cnpq.br/6759395798493082Souza, Joelma Rodrigues dehttp://lattes.cnpq.br/6624702445172439Neves, Adriana Freitashttp://lattes.cnpq.br/2984939300978146Goulart, Vivian Alonsohttp://lattes.cnpq.br/6846069044649305Vecchi, Larahttp://lattes.cnpq.br/9476822610412231Lopes, Patrícia da Silva2021-01-22T11:49:28Z2021-01-22T11:49:28Z2020-05-29info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfLOPES, Patrícia da Silva. Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue. 2020. 95 f. Tese (Doutorado em Ciências da Saúde)- Universidade Federal de Uberlândia, Uberlândia, 2020. DOI http://doi.org/10.14393/ufu.te.2020.3021https://repositorio.ufu.br/handle/123456789/31127http://doi.org/10.14393/ufu.te.2020.3021porinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFUinstname:Universidade Federal de Uberlândia (UFU)instacron:UFU2022-07-27T18:20:23Zoai:repositorio.ufu.br:123456789/31127Repositório InstitucionalONGhttp://repositorio.ufu.br/oai/requestdiinf@dirbi.ufu.bropendoar:2022-07-27T18:20:23Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU)false
dc.title.none.fl_str_mv Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue
Development of molecules with teranostic potential for zika and dengue viruses
title Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue
spellingShingle Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue
Lopes, Patrícia da Silva
Phage-Display
Phage-Display
vírus Dengue
Dengue virus
vírus Zika
Zika virus
Inibição
Inhibition
Diagnóstico
Diagnostic
CNPQ::CIENCIAS DA SAUDE
Vírus da Zika
Dengue
title_short Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue
title_full Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue
title_fullStr Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue
title_full_unstemmed Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue
title_sort Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue
author Lopes, Patrícia da Silva
author_facet Lopes, Patrícia da Silva
author_role author
dc.contributor.none.fl_str_mv Santos, Paula Souza
http://lattes.cnpq.br/5065438852500935
Goulart Filho, Luiz Ricardo
http://lattes.cnpq.br/6759395798493082
Souza, Joelma Rodrigues de
http://lattes.cnpq.br/6624702445172439
Neves, Adriana Freitas
http://lattes.cnpq.br/2984939300978146
Goulart, Vivian Alonso
http://lattes.cnpq.br/6846069044649305
Vecchi, Lara
http://lattes.cnpq.br/9476822610412231
dc.contributor.author.fl_str_mv Lopes, Patrícia da Silva
dc.subject.por.fl_str_mv Phage-Display
Phage-Display
vírus Dengue
Dengue virus
vírus Zika
Zika virus
Inibição
Inhibition
Diagnóstico
Diagnostic
CNPQ::CIENCIAS DA SAUDE
Vírus da Zika
Dengue
topic Phage-Display
Phage-Display
vírus Dengue
Dengue virus
vírus Zika
Zika virus
Inibição
Inhibition
Diagnóstico
Diagnostic
CNPQ::CIENCIAS DA SAUDE
Vírus da Zika
Dengue
description Arboviruses caused by the Dengue virus (DENV) and Zika (ZIKV), are currently considered diseases that represent a potential challenge to public health, resulting in intense and limiting clinical manifestations, which can culminate in microcephaly (ZIKV) and death. There are still no vaccines in the public network or any other preventive drug, the diagnosis is inaccurate due to the cross reactions between the antibodies of DENV, ZIKV and other flaviviruses. Given this context, we aim to develop new molecules based on small peptides (phage) that bind to the ZIKV and DENV-2 virus using the Phage-Display methodology. Thus, the intention of the present study was to select, characterize and validate ZIKV and DENV-2 ligands, assessing their ability to inhibit and also differentiate ZIKV and DENV. The ELISA immunoassay, ZIKV and DENV-2 entry inhibition assay in vitro by flow cytometry using Vero cells and 4G2 antibody and silica analysis were used for the validation of the selected phages. ELISA was also performed with a phage that did not show significant viral inhibition and another that was effective for inhibition using samples from patients infected with DENV and ZIKV. Phage D4, selected for ZIKV, was able to inhibit the entry of ZIKV and DENV-2. F12 phage, also selected for ZIKV, was not able to inhibit ZIKV entry, however it was able to discriminate DENV patients from ZIKV patients and healthy individuals (p <0.0001) with high accuracy, presenting 94.9% specificity and 81.2% sensitivity for DENV infection. The G8 phage selected for DENV-2 was able to inhibit the entry of DENV-2 and also discriminated against DENV from ZIKV and healthy patients in a preliminary test that will be optimized with a larger number of samples. The silica study showed that the possible interaction sites of the peptides expressed by the D4 phage, which inhibit the entry of ZIKV and DENV, were located in domain III of the envelope protein (EDIII) or very close to the fusion loop. Both EDIII and the fusion loop, which is located in EDII, are important target epitopes for antibodies already described in the literature with wide neutralizing action for flavivirus. In the study of phages F12 and G8, which differentiated DENV patients from ZIKV and healthy individuals, we observed their interactions with amino acid residues from the ZIKV and DENV-2 envelope protein, which comprise the kl loop, which is discussed in comparative studies of Zika and Dengue viruses. Through these studies we will seek the integration of important methodologies, covering low cost, speed and high efficiency, in order to fill possible gaps and demands that we still find in the diagnosis of DENV and ZIKV in different stages of the infection, as well as to contribute to future complementary studies, in order to produce possible therapeutic and preventive interventions. In general, this study demonstrated that Phage-Display technology is a strong tool for the development of molecules with antiviral and diagnostic potential. With this, the next step will be to synthesize the peptides expressed in the phages, carrying out tests that can contribute to the development of vaccines for ZIKV, DENV or even for other infectious agents from synthetic molecules.
publishDate 2020
dc.date.none.fl_str_mv 2020-05-29
2021-01-22T11:49:28Z
2021-01-22T11:49:28Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
format doctoralThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv LOPES, Patrícia da Silva. Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue. 2020. 95 f. Tese (Doutorado em Ciências da Saúde)- Universidade Federal de Uberlândia, Uberlândia, 2020. DOI http://doi.org/10.14393/ufu.te.2020.3021
https://repositorio.ufu.br/handle/123456789/31127
http://doi.org/10.14393/ufu.te.2020.3021
identifier_str_mv LOPES, Patrícia da Silva. Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue. 2020. 95 f. Tese (Doutorado em Ciências da Saúde)- Universidade Federal de Uberlândia, Uberlândia, 2020. DOI http://doi.org/10.14393/ufu.te.2020.3021
url https://repositorio.ufu.br/handle/123456789/31127
http://doi.org/10.14393/ufu.te.2020.3021
dc.language.iso.fl_str_mv por
language por
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universidade Federal de Uberlândia
Brasil
Programa de Pós-graduação em Ciências da Saúde
publisher.none.fl_str_mv Universidade Federal de Uberlândia
Brasil
Programa de Pós-graduação em Ciências da Saúde
dc.source.none.fl_str_mv reponame:Repositório Institucional da UFU
instname:Universidade Federal de Uberlândia (UFU)
instacron:UFU
instname_str Universidade Federal de Uberlândia (UFU)
instacron_str UFU
institution UFU
reponame_str Repositório Institucional da UFU
collection Repositório Institucional da UFU
repository.name.fl_str_mv Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU)
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