Affinity enhancement of CR3022 binding to RBD; in silico site directed mutagenesis using molecular dynamics simulation approaches.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a disease which caused by a novel beta coronavirus. Structural and non-structural proteins are expressed by the virus gene fragments. The RBD of the S1 protein of the virus has the ability to interact with potent antibodies including CR3022, which was characterized to target the S protein of the virus which can efficiently neutralize the SARS-CoV in vitro and in vivo. In current study, we aimed to design CR3022 based antibody with high affinity compared with wild-type CR3022 using MD simulation method. Two variants were designed based on the amino acid binding conformation and the free binding energy of the critical amino acids which involved in CR3022-RBD interactions were evaluated. In this study three complexes were evaluated; CR3022-RBD, V1-RBD and V2-RBD using molecular dynamics simulations carried out for 100 ns in each case. Then, all the complexes were simulated for 100 ns. In the next step, to calculate the free binding affinity of the wild CR3022 and mutant antibody (V1 and V2) with RBD, the PMF method was performed. The RMSD profile demonstrated that all three complexes were equilibrated after 85 ns. Furthermore, the free binding energy results indicated that the V2-RBD complex has the higher binding affinity than V1-RBD and CR3022-RBD complexes. It should be noted that in above variants, the electrostatic energy and the number of H-bonds between the antibody and RBD increased. Thus, it is suggested that both designed antibodies could be considered as appropriate candidates for covid-19 disease treatment.Communicated by Ramaswamy H. Sarma.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

Interaction of Human ACE2 to Membrane-Bound SARS-CoV-1 and SARS-CoV-2 S Glycoproteins.

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is responsible for the current global coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The viral entry of SARS-CoV-2 depends on an interaction between the receptor-binding domain of its trimeric spike glycoprotein and the human angiotensin-converting enzyme 2 (ACE2) receptor. A better understanding of the spike/ACE2 interaction is still required to design anti-SARS-CoV-2 therapeutics. Here, we investigated the degree of cooperativity of ACE2 within both the SARS-CoV-2 and the closely related SARS-CoV-1 membrane-bound S glycoproteins. We show that there exist differential inter-protomer conformational transitions between both spike trimers. Interestingly, the SARS-CoV-2 spike exhibits a positive cooperativity for monomeric soluble ACE2 binding when compared to the SARS-CoV-1 spike, which might have more structural restraints. Our findings can be of importance in the development of therapeutics that block the spike/ACE2 interaction.