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Doctoral Thesis
DOI
https://doi.org/10.11606/T.42.2021.tde-21012022-122317
Document
Author
Full name
Robert Andreata Santos
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2021
Supervisor
Committee
Ferreira, Luis Carlos de Souza (President)
Araujo, Ana Priscila Perini de
Durigon, Edison Luiz
Granato, Celso Francisco Hernandes
Title in Portuguese
Desenvolvimento de plataformas de diagnóstico uniplex e multiplex para infecção com arbovírus utilizando proteínas recombinantes.
Keywords in Portuguese
Chikungunya
Dengue
Diagnóstico Sorológico
Epidemiologia
Proteínas recombinantes
Zika
Abstract in Portuguese
Os vírus Dengue (DENV), Zika (ZIKV) e Chikungunya (CHIKV) são arbovírus evidentes no cenário internacional atual que apresentam dificuldade na geração de dados epidemiológicos acurados. Tal fato ocorre devido à grande semelhança dos sintomas clínicos compartilhados pelas doenças, além da elevada reação cruzada observada entre alguns desses vírus quando avaliados em testes de diagnóstico sorológico. A disponibilidade de testes sorológicos sensíveis, específicos e de baixo custo contra esses vírus ainda é limitada no país. Diante disso, o presente projeto teve por objetivo desenvolver plataformas de diagnóstico sorológico uniplex (ELISA) e multiplex (Luminex), baseadas em formas recombinantes de proteínas DENV, ZIKV e CHIKV. Após avaliação in silico, porções da proteína não estrutural 1 (NS1) de DENV e ZIKV (denominadas ΔNS1 ZIKV e ΔNS1 DENV1-4), e da proteína 2 do envelope (E2) de CHIKV (denominada E2.1 CHIKV) apresentaram baixa homologia entre si e com as mesmas regiões de vírus próximos a DENV, ZIKV e CHIKV, sendo escolhidas como antígenos de diagnóstico. As proteínas foram implementadas nas plataformas de sorodiagnóstico, as quais foram otimizadas e validadas com a utilização de painéis de soros humanos fornecidos por colabores. Todos os testes gerados na plataforma de ELISA apresentaram valores de sensibilidade e especificidade iguais ou superiores aos testes atualmente disponíveis no mercado, com destaque para o teste ELISA ZIKV, o qual proporcionou uma parceria público privada que culminou na aprovação da sua comercialização pela ANVISA. Resultados similares foram observados com a implementação dos antígenos à plataforma Luminex, corroborando o potencial dos antígenos desenvolvidos. O sucesso do desenvolvimento do presente projeto salienta a importância do estudo e do desenvolvimento de metodologias diagnósticas, bem como ilustra de forma clara como a pesquisa acadêmica pode ser feita em sintonia com empresas voltadas para o desenvolvimento de tecnologias na área da saúde.
Title in English
Development of uniplex and multiplex diagnostic platforms for arbovirus infection using recombinant proteins.
Keywords in English
Chikungunya
Dengue
Epidemiology
Recombinant proteins
Serological diagnosis
Zika
Abstract in English
The Dengue (DENV), Zika (ZIKV) and Chikungunya (CHIKV) viruses are arboviruses evident in the current international scenario that present difficulties in the generation of accurate epidemiological data. This fact occurs due to the great similarity among the clinical symptoms shared by the diseases, in addition to the high cross reaction observed among some of these viruses when evaluated by serological diagnostic tests. The availability of sensitive, specific and low-cost serological tests against these viruses is still limited in Brazil. Therefore, this project aimed to develop uniplex (ELISA) and multiplex (Luminex) serological diagnosis platforms, based on recombinant forms of DENV, ZIKV and CHIKV proteins. After in silico evaluation, portions of the non-structural protein 1 (NS1) of DENV and ZIKV (called ΔNS1 ZIKV and ΔNS1 DENV1-4), and of the envelope protein 2 (E2) of CHIKV (called E2.1 CHIKV) presented low homology among themselves and with the same regions of viruses close to DENV, ZIKV and CHIKV, being chosen as diagnostic antigens. The proteins were implemented on the serodiagnosis platforms, which were optimized and validated using human serum panels provided by collaborators. All tests generated on the ELISA platform showed sensitivity and specificity values equal to or higher than the tests currently available on the market, with emphasis on the ELISA ZIKV test, which provided a public-private partnership that culminated in the approval of its commercialization by ANVISA. Similar results were observed with the implementation of the antigens to the Luminex platform, corroborating the potential of the developed antigens. The success of the development of this project highlights the importance of studying and developing diagnostic methodologies, as well as illustrate clearly how academic research can be done in tune with companies focused on the development of technologies in the health area.
 
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Publishing Date
2022-12-02
 
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