Functional Study Of Neutralizing Nanobodies Against Novel Coronavirus(SARS-CoV-2)

The novel coronavirus(Severe acute respiratory syndrome coronavirus 2,SARSCoV-2)caused a global pandemic of COVID-19(Coronavirus disease 2019),bringing a devastating global health crisis to the world.Neutralizing antibodies are one of the effective antiviral countermeasures because they can block the infection of normal cells by the SARS-CoV-2.Due to their unique structure and functional activity,shark nanobodies have shown their huge application space in the field of biomedicine in recent years.Searching for new crown neutralizing antibodies in shark nanobodies may provide a new idea for the treatment of COVID-19.In this study,a series of research work was carried out on the anti-SARS-CoV-2 shark nanobodies previous found in the laboratory in terms of biological activity,broad spectrum,stability,etc,and explored the mechanism of Omicron(B.1.1.529)mutant escaping antibody neutralization.This study provide a new idea for the development of broadly neutralizing antibodies or vaccines in the future.The main works conducted in the article are summarized as follows:1.The expression of the monovalent shark nanobody 20G6 and the bivalent shark nanobody 20G6-Fc in different expression systems,and the proteins were purified using different purification methods,refer to yield and purity,activity,production process and cost,we choose Escherichia coli to express monovalent 20G6,and mammalian cells to express bivalent 20G6-Fc,and further purified and concentrated the antibodies for subsequent experiments.2.Through enzyme-linked immunosorbent assay technology,biofilm interference technology,and new coronavirus pseudovirus detection system,the binding activity and neutralization activity of shark nanobodies to the SARS-CoV-2 wild type and mutants were detected,and it was found that 20G6 can effectively neutralize SARSCoV-2 WH and its mutants.3.Detect the biological activity of shark nanobody 20G6 on Sarbecovirus subgenus SARS-related coronaviruses,explore the broad-spectrum neutralizing potency of 20G6,and found that it could neutralize pangolin-CoV(pangolin coronavirus)Bat-CoV(bat coronavirus)in the Sarbecoviruses family,with a certain broad-spectrum neutralizing potency.4.To study the stability of shark nanobodies,including thermal stability and acid resistance,we use ELISA to detect the binding activity between antibodies and antigens.We found that whether 20G6 was treated in a high temperature with 90℃ for 1 hour,or stored in a p H 2 solution for three weeks,it could still maintain strong antigenbinding activity,which showed that the shark nanobody 20G6 has good tolerance to acidic environment and high thermal stability.5.Detect the functional activity of 20G6 on the SARS-CoV-2 Omicron variant,and the results showed that 20G6 does not bind to Omicron and cannot neutralize the Omicron pseudovirus.Combined with the research foundation of epitope structure analysis in the laboratory,and analyzed the amino acid mutation sites of mutant strains,the influence of a single important mutated site on antigen-antibody binding activity was explored,and it was found that the escape of Omicron from 20G6 neutralization may mainly depend on the S375F mutation.