Expression and characterization of single-chain variable fragments (scFv) intrinsically fluorescents for SARS-COV-2 detection.

• Grant number: 23/08872-9
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: August 01, 2023
• End date: July 31, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Grantee: Marcelo Bergamin Zani
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 21/11936-3 - Center for Translational Science and Biopharmaceutical Development, AP.CCD

Abstract

Coronavirus is a family of viruses composed of a wide variety of species capable of infecting different animals. Among them, SARS-CoV-2 is the species responsible for the COVID-19 disease, which generated the entire global pandemic scenario we are currently experiencing. The spike (S) protein has been one of the main targets of the scientific community due to its essential role in viral infection. This protein interacts with the cell membrane receptor ACE2 and culminates in the cell internalization of the virus, mediated by the fusion of the viral envelope with the plasmatic membrane of the host cell, thus initiating the viral replication cycle. ACE2 receptors are highly expressed in the respiratory system, which gives the virus its capacity for air transmission, but they are also expressed in other regions of the human body, such as the kidneys, heart, intestines and endothelium of organs, justifying the occurrence of other symptoms in addition to respiratory disorders also observed in the disease. Mutations in S were responsible for the appearance of new variants of SARS-CoV-2. Another protein that has been identified as an important target of study is the N protein (N), whose units involve the viral RNA (nucleocapsid). Both protein S and protein N perform essential functions for SARS-CoV-2, making them potential study targets for the development of vaccines and drugs to combat the disease. One of the advantages of the N protein, as a target, in relation to the S protein is that it is more conserved and stable, as it presented fewer mutations over time.Recombinant antibodies constitute the most expanding class of drugs and have generated new highly effective treatments, which can be selected via phage display technology. Recombinant antibodies can be expressed in different formats, one of them being the scFv format, formed by the fusion of the variable domains of the light and heavy chain of an immunoglobulin through a polypeptide sequence called linker. Unlike the other types, scFv molecules do not require post-translational modifications and can be studied in prokaryotic expression systems. These modified antibodies, equally capable of binding to antigens, can be produced for various purposes, for example, as diagnostic tools for detecting antigens in samples. Additionally, fluorescent recombinant proteins can be obtained through the use of so-called orthogonal pairs (tRNA-tRNA synthetases) which, when co-expressed with a recombinant protein, insert a non-canonical chromophore amino acid in the amber codon (UAG) of this sequence.This research aims to produce fluorescent anti-Spike and anti-protein N scFvs. For this, we intend to use the orthogonal-pair technology to insert the non-canonical amino acids p-azido-phenylalanine (pAzF) and AA-coumarin, in the suppression of the amber codon in the expression of scFvs selected by phage display. Obtaining anti-S and/or anti-N scFvs labeled with AA-coumarin residues (fluorescent), for example, can simplify assays based on the immunodetection of SARS-COV-2 in biological samples (Ex: rapid assay lateral flow).