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Application of natural SARS-CoV-2 proteins to aid the development of new diagnostic strategies for COVID-19


COVID-19 represents an unprecedented pandemic and tends to become endemic and recurrent due to the characteristics of the SARS-CoV-2 virus and its mutations. With the continuous increase in the number of cases worldwide, the lack of vaccines or effective treatments, the health system is significantly impacted by the high demand for resources to diagnose and treat patients. To understand the infection and fight the pandemic, detailed characterization of SARS-CoV-2 structural proteins primary structure, post-translational modifications, interactions and antigenicity are essential. Pathophysiology and virulence mechanisms of SARS-CoV-2 have been associated with the structural elements, spike (S), membrane protein (M) and envelope (E) proteins. Particularly the glycoprotein S plays important role in viral attachment to the ACE2 receptor in the surface of the host cell, fusion and entry, process depend on specific post-translational modifications to promote virulence. These characteristics in S protein and potentially in other structural proteins are mediated by the constant mutations in viral RNA. These mutations and resulting pos-translational modifications also produce cleavage sites and neo-epitopes that have immediate applications to improve the specificity and sensibility of diagnostics approaches. Here, we seek to explore a combination of biochemical approaches to characterize structural proteins of SARS-CoV-2 as well as its interactions with host cells. After identification and selection of important regions on surface and membrane proteins, we will develop affinity agents and mass spectrometric based approaches to detect and quantify the virus with high sensitivity and specificity. To achieve this general goal, we propose: i) to characterize in detail by mass spectrometry the natural viral proteins obtained from patient samples and recombinant proteins submitted to infection models, with a focus on possible post-translational mutations or modifications; ii) express and purify important domains of SARS-CoV-2 Spike (S), E (envelope) and M (membrane) proteins; iii) identify and synthesize peptides that represent important antigens and are responsible for interacting regions with host cell proteins; iv) produce synthetic heavy variable chain antibodies using phage libraries against natural proteins, expressed domains and selected synthetic peptides; v) scale-up the production of recombinant proteins, synthetic antibodies and peptides and conjugate them with particles to allow a wide exploration of interactions, as well as multiplex diagnostic platforms and rapid assays. Inevitably, developments from this project may also translate into potential therapeutic molecules or vaccine candidates, based on their cell infection neutralizing capabilities or high antigenicity response. More importantly, with a strong collaborative mindset, we will make available these scaled-up molecules for parallel initiatives focused on COVID19 diagnostics, therapy or vaccines, in order to speed up national technological developments and promote, at a least in part, independence from international markets. In summary, the combination of local expertise and a network of specialists and relevant clinical samples, we strongly believe that this effort greatly increases the chances of providing timely and successful answers to the demands in diagnostic of COVID19 in a moment of limited resources and great suffering worldwide. (AU)

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(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
NASCIMENTO, EVAIR D.; FONSECA, WILSON T.; DE OLIVEIRA, TASSIA R.; DE CORREIA, CAMILA R. S. T. B.; FACA, VITOR M.; DE MORAIS, BEATRIZ P.; SILVESTRINI, VIRGINIA C.; POTT-JUNIOR, HENRIQUE; TEIXEIRA, FELIPE R.; FARIA, RONALDO C.. COVID-19 diagnosis by SARS-CoV-2 Spike protein detection in saliva using an ultrasensitive magneto-assay based on disposable electrochemical sensor. SENSORS AND ACTUATORS B-CHEMICAL, v. 353, p. 9-pg., . (19/03943-0, 16/25798-3, 20/12920-0, 20/04635-4, 20/07539-6)

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