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Flávio Guimarães da Fonsecahttp://lattes.cnpq.br/4028759481820525Bárbara Resende Quinanhttp://lattes.cnpq.br/2743695327107987Erna Geessien KroonBeatriz Senra Álvares da Silva SantosCíntia Lopes de Brito MagalhãesMarcele Neves Rochahttp://lattes.cnpq.br/2498047677232858Sabrynna Brito Oliveira2019-11-05T14:03:47Z2019-11-05T14:03:47Z2019-09-30http://hdl.handle.net/1843/30833https://orcid.org/0000-0001-9303-4338A dengue é uma das principais arboviroses que afeta o ser humano e um grave problema de saúde pública. Os quatro sorotipos do Dengue virus (DENV1, 2, 3 e 4) são transmitidos através da picada de mosquitos fêmea do gênero Aedes. A vacina disponível atualmente apresenta lacunas imunológicas, o que motiva a continuidade de pesquisas em busca de uma vacina eficaz. O vírus Vaccinia Ankara Modificado (MVA) está entre os vetores virais mais promissores e intensamente estudados mundialmente e é considerado como uma excelente ferramenta para a geração de vacinas. O objetivo dessa pesquisa foi construir MVA recombinantes expressando a proteína E de DENV1, 2 e 4 e avaliar o potencial protetor dessas construções em modelo murino. Genes sintéticos da proteína E de DENV1, 2 e 4 foram desenhados, otimizados, obtidos comercialmente e subclonados no plasmídeo pLW44, dando origem aos plasmídeos de transferência, utilizados na construção dos vírus recombinantes. Culturas de fibroblasto de embrião de galinha (CEF) ou BHK-21 foram infectadas por MVA e transfectadas pelos plasmídeos de transferência pLW44-DENV1, pLW44-DENV2 ou pLW44-DENV4, separadamente, para a construção dos MVAs recombinantes (rMVAs). Os rMVAs gerados tiveram a sua construção confirmada por PCR, sequenciamento e citometria de fluxo. O potencial protetor do rMVA-DENV1 foi testado em camundongos C57/BL6 em esquema de imunização dose-reforço homólogo, seguido de desafio intracranial com o DENV correspondente. Os animais foram imunizados com 107 pfu ou 108 pfu de rMVA-DENV1 e desafiados com DENV1 Mochizucki. Durante o desafio os animais foram monitorados e avaliados quanto ao seu estado geral de saúde pelo protocolo SHIRPA. Com base nos resultados obtidos podemos concluir que as construções dos diferentes vírus recombinantes se deram de maneira eficaz, sobretudo a de rMVA-DENV1, a única testada quanto à capacidade protetora. Os dados gerados nessa pesquisa serão somados aos obtidos anteriormente para DENV3 visando à obtenção de uma vacina tetravalente contra DENV.Dengue is the main arbovirus that affecting humans and a serious public health problem. The four dengue virus serotypes (DENV1, 2, 3 and 4) are transmitted through the bite of female Aedes mosquitoes. A vaccine available worldwide has immunological gaps, which motivates further research for an effective vaccine. The Modified Vaccinia Ankara Virus (MVA) is among the most advanced and intensely studied viral vectors worldwide, and is considered as an excellent tool for vaccine generation. The aim of this work was to construct recombinant MVAs expressing the protein E of DENV1, 2 and 4 and to evaluate the protective potential of these constructs in a murine model. Synthetic genes coding for protein E from DENV1, 2 and 4 were designed, optimized, commercially obtained and subcloned into plasmid pLW44, giving rise to transfer plasmids used in the construction of recombinant viruses. Chicken embryo fibroblast cultures – CEF, or BHK-21 were infected with MVA and transfected with the transfer plasmids pLW44-DENV1, pLW44-DENV2 and pLW44-DENV4 for the construction of recombinant MVAs (rMVA). The generated rMVAs had their correct construction confirmed by PCR, sequencing and flow cytometry. The protective potential of rMVA-DENV1 was tested in C57 / BL6 mice on a homologous dose-boosting immunization schedule, followed by intracranial challenge with the corresponding DENV. Animals were immunized with 107 pfu or 108 pfu of rVA-DENV1 and challenged with DENV1 Mochizuki. During the challenge, they were monitored evaluated through a SHIRPA protocol. Based on the results we can conclude that the construction of the different recombinant viruses was effective, especially rMVA-DENV1, the only one tested for protective capacity. The data generated in this research can be added to those previously obtained for DENV3 in order to obtain a tetravalent vaccine against DENV.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorporUniversidade Federal de Minas GeraisPrograma de Pós-Graduação em MicrobiologiaUFMGBrasilICB - INSTITUTO DE CIÊNCIAS BIOLOGICAShttp://creativecommons.org/licenses/by-nc-nd/3.0/pt/info:eu-repo/semantics/openAccessMicrobiologiaVírus da DengueVacinas sintéticasVírus Vacciniavírus da denguevacina recombinanteMVAConstrução e avaliação do potencial protetor, em modelo murino, de diferentes construções do Vaccinia Virus Ankara Modificado (MVA) expressando a proteína E de Dengue Virus sorotipos 1, 2 e 4.info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMGORIGINALTESE_SABRYNNA_2019.pdfTESE_SABRYNNA_2019.pdfapplication/pdf5207163https://repositorio.ufmg.br/bitstream/1843/30833/1/TESE_SABRYNNA_2019.pdff0753668287d8f26a4e360eef490fe43MD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufmg.br/bitstream/1843/30833/2/license_rdfcfd6801dba008cb6adbd9838b81582abMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82119https://repositorio.ufmg.br/bitstream/1843/30833/3/license.txt34badce4be7e31e3adb4575ae96af679MD53TEXTTESE_SABRYNNA_2019.pdf.txtTESE_SABRYNNA_2019.pdf.txtExtracted texttext/plain246682https://repositorio.ufmg.br/bitstream/1843/30833/4/TESE_SABRYNNA_2019.pdf.txt0d9f12a9af5702b0620565659473e770MD541843/308332019-11-14 12:54:13.217oai:repositorio.ufmg.br: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Repositório InstitucionalPUBhttps://repositorio.ufmg.br/oaiopendoar:2019-11-14T15:54:13Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
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