Affinity maturation of single-chain antibodies against SARS-CoV-2 receptor binding domain and yeast surface display tracking

Abstract

The new coronavirus, the cause of COVID-19, is a global threat to human health. The virus, called SARS-CoV-2, has a high transmissibility and mortality rate. In addition, there is still a growing concern about the new variants: alpha, beta, gamma, and delta, which have a higher rate of contagion and mortality and are more resistant to vaccines and medicines used. Furthermore, characteristics of the Brazilian's reality represent an aggravating factor for the pandemic, mainly due to the delay in obtaining sufficient vaccination and testing coverage to curb the virus's contamination, among others. Thus, it becomes increasingly urgent and essential to develop and apply fast, low-cost, and easy-to-apply diagnostic tests that are specific, sensitive to new variants, and that present high affinity to the new coronavirus. Therefore, the objective of this project is to determine in vitro the affinity maturation of the single-chain variable fragment (scFv) of the CC12.1 antibody with the receptor-binding domain (RBD) of the spike protein of SARS-CoV- 2, through in silico tools and Yeast Surface Display (YSD) technique. In order to determine the amino acids to be mutated, different in silico analyzes will be performed to assess the antigen and antibody interaction. Subsequently, the residues will be mutated by site-directed PCR. Plasmids containing the scFv coding sequence and mutants will be transformed into Saccharomyces cerevisiae, induced and the interaction will be tracked by evaluating the affinity of the antibody located on the yeast surface with the antigen by flow cytometry (YSD). In this way, a study aimed at increasing the affinity of the variable fragment will be carried out, leading to the selection of a reagent with greater specificity and sensitivity to SARS-CoV-2. (AU)