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Development of uniplex and multiplex diagnostic platforms for arbovirus infection using recombinant proteins

Grant number: 19/16049-5
Support type:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): November 11, 2019
Effective date (End): November 10, 2020
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal researcher:Luis Carlos de Souza Ferreira
Grantee:Robert Andreata Santos
Supervisor abroad: Jean-Claude Manuguerra
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Research place: Institut Pasteur, France  
Associated to the scholarship:16/23560-0 - Development of uniplex and multiplex diagnostic platforms for arbovirus infection using recombinant proteins, BP.DR


The aim of the main research project related to the present proposal is to develop and validate low-cost specific diagnosis platforms, based on recombinant proteins, and designed for uniplex and multiplex detections of arboviral infections. To achieve the proposed aim, the NS1 proteins of DENV and ZIKV were targeted, since these were normally used in DENV antibody detection. Our results demonstrated that the recombinant antigen spanning the C-terminus of NS1 protein preserves antigenicity and concentrates the majority of conformational epitopes that confer specificity to the antibodies generated during infection, since similar reactivity was found when compared to the whole and truncated versions of protein (patent number: BR102016011318-0). This is supported by the identification of conformational epitopes present in the protein, capable of being recognized by B cells of the immune system. However, a high recognition of DENV positive samples can be observed when evaluating the entire NS1 protein, with a drastic reduction in the recognition of the same sera when evaluating the ”NS1 protein. Furthermore, as demonstrated by Song and colleagues, the region encompassing the ”NS1 protein has structural potential and electrostatic surface charges capable of promoting differentiation between antibodies directed against DENV or ZIKV (SONG et al., 2016).The visit at Dr. Jean-Claude Manuguerra's laboratory aimed the standardization of the test using the ZIKV and DENV1-4 proteins into the multiplex diagnostic platform previously described by the CIBU (CAO-LORMEAU et al., 2016; AUBRY et al., 2017). The preliminary characterization faced difficulties predicted by the standardized ELISA test, since the C-terminus of the NS1 protein drastically reduces cross-reactivity but does not extinguish it, and the detection of reactivity in the Luminex assay is much more sensitive than in the ELISA tests. Thus, cross-reaction between DENV positive samples and ZIKV ”NS1 protein was observed, but not in the opposite sense, since the ZIKV-positive samples tested were only positive for ZIKV.As an attempt to detect antibodies present under conditions of acute infections, the presence of specific IgG3 was evaluated in the samples, as demonstrated in a comparative epidemiological study between DENV and ZIKV (RODRIGUEZ-BARRAQUER et al., 2019). Classical detection of acute phase diseases is confirmed by the presence of specific IgM antibodies in the serum of suspected patients due to DENV or ZIKV infection. However, such detection is not always possible to be performed in a specific and sensitive way, since inactivation and / or depletion of IgG render the tests unviable. The results showed the capacity of detecting IgG3 in acute samples for ZIKV infection. A greater follow up of each case is necessary for the evaluation of the increase of reactivity after infection, as well as the its reduction after 6 months.Aiming to further develop the multiplex assay generated with the ”NS1 proteins, as well as insert the E2 CHIV protein in the Luminex detection, we contacted Dr. Jean-Claude Manuguerra's laboratory to propose a long stay at the CIBU unit. We expect that our project's antigens will successfully be standardized at the Luminex platform with the capacity of differentiate between different arboviruses infection in a single test. For all these reasons we believe that the required fellowship will be translated to significant advances in the field, allowing further prospective and retrospective epidemiological arboviral studies, as well as the monitoring of probable outbreaks.

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