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Expression and analysis of recombinant structural proteins of dengue-2 and yellow fever viruses produced by baculovirus

Grant number: 12/23522-0
Support Opportunities:Regular Research Grants
Duration: March 01, 2013 - February 28, 2015
Field of knowledge:Health Sciences - Medicine - Medical Clinics
Principal Investigator:Benedito Antônio Lopes da Fonseca
Grantee:Benedito Antônio Lopes da Fonseca
Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil


The dengue (DENV) and yellow fever (YFV) viruses are the main arboviruses (arthropod-borne viruses) circulating in Brazil. They are transmitted by mosquitoes of the genus Aedes and cause the diseases called dengue and yellow fever, respectively. Dengue is currently the human disease transmitted by an arbovirus of greatest significance to public health worldwide. These viruses are prevalent mainly in countries located on the tropical and subtropical regions of the world, with an estimate of approximately 50 to 100 million new cases of dengue per year. Yellow fever is prevalent in the tropical regions of the Americas and Africa and it is responsible for sporadic human cases, usually with greater severity than dengue. The four dengue virus serotypes are called dengue-1, -2, -3 and -4 (DENV-1 to -4) while the yellow fever virus has only one dominant serotype. Dengue presents as infections ranging from asymptomatic infections to extremely severe clinical conditions characterized hypotension, bleeding, and varying degrees of circulatory shock, in addition to no less severe manifestations, characterized by neurological impairment, hepatic and myocardial involvement. To date, there is neither a specific drug treatment for dengue nor a vaccine that protects against infection by these viruses. Yellow fever also has a range of clinical presentations, whose most severe manifestations are characterized by generalized bleeding and liver failure, a disease with a high mortality rate. In contrast, there is an extremely immunogenic vaccine against yellow fever virus that, in a small percentage of cases, presents an exacerbated reactogenicity, resulting in a serious adverse event characterized by an important viscerotropism and high fatality rate. For these reasons, with this study design, we are aiming at the production of recombinant proteins of these viruses in a eukaryotic system, so that the surface of the envelope glycoproteins of DENV-2 and YFV will undergo a post-translational processing close to that observed with wild-type virus infection and expressed as recombinant proteins that are recognized by the immune system as the natural structural proteins of these two viruses. This study design is intended to produce the proteins of these two viruses as a prototype and, if we succeed, we will try to express the structural proteins of other DENV serotypes and use them either in diagnostic methods or to produce a tetravalent vaccine candidate against dengue virus. This is a collaboration project with Professor Yorgo Modis, of Yale University, where one of the participating students will spend sometime to learn how to solve possible problems encountered when using the technique or expression of recombinant proteins in baculovirus. The genes of structural proteins prM/E will be PCR amplified, cloned and transfected into baculovirus, and then expressed in Sf9 cells for production of recombinant proteins. After verification of the proper expression of these proteins, they will be inoculated in mice and the immune response elicited against the structural proteins of the wild dengue virus and yellow fever will be assessed. If immunogenic, the same strategy will be used to express the structural proteins of all other three dengue serotypes. (AU)

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