key: cord-0699316-vyjnagtm authors: Hayashi, Takuma; Yaegashi, Nobuo; Konishi, Ikuo title: Effect of RBD mutation (Y453F) in spike glycoprotein of SARS-CoV-2 on neutralizing antibody affinity date: 2020-11-28 journal: bioRxiv DOI: 10.1101/2020.11.27.401893 sha: 76fa02e5314a308f2ef8e057cef1d59ef3d92eb1 doc_id: 699316 cord_uid: vyjnagtm Natural selection “adaptation” in the coronavirus can occur during coronavirus amplification in vivo in farmed minks. Natural selection in such viruses is observed by introduction of mutations in SARS- CoV-2 that are not observed during the growth process in humans. Infection with a mutant (Y453F) of SARS-CoV-2 from farmed minks is known to widely spread among humans. We investigated the virological characteristics of this SARS-CoV-2 mutant (Y453F) using three-dimensional protein structural analysis. Our experimental study suggests that virus variants with the Y453F mutation partially escaped detection by four neutralizing monoclonal antibodies. The spread of SARS-CoV-2 variants mediated by millions of infected farmed minks is uncontrolled; consequently, raising a concern that infection of SARS-CoV-2 mutants that cause serious symptoms in humans may spread globally. Mutations in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can jeopardize the efficacy of potential vaccines and therapeutics against coronavirus disease-2019 (COVID-19). Animals of the Mustelidae family, such as minks and ferrets can be infected with SARS-CoV-2 relatively easily compared with other mammals (1) . However, the reason why SARS-CoV-2 is extremely contagious to these animals remains to be elucidated. Nonetheless, it is clear that when several farmed minks kept in a high-density environment are infected with SARS-CoV-2, the virus proliferates in large numbers. Consequently, humans and minks may be at high risk of SARS-CoV-2 infection. Natural selection "adaptation" in the coronavirus can occur during coronavirus amplification in vivo in farmed minks (2) . Natural selection in such viruses is observed by introduction of mutations in SARS-CoV-2 that are not observed during the growth process in humans (2, 3) . Infection with a mutant (Y453F) of SARS-CoV-2 from farmed minks is known to widely spread among humans (4). We investigated the virological characteristics of this SARS-CoV-2 mutant (Y453F) using threedimensional protein structural analysis. SARS-CoV-2 mutant has an amino acid mutation Y453F in the sequence encoding spike glycoprotein (4) . This SARS-CoV-2 mutant has been detected in approximately 300 viral sequences isolated from the European and Dutch population as well as in minks. Data on the three-dimensional structure of the receptor binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 (PDB ENTITY SEQ 6VW1_1) was used in these studies (5) . Data (PDB: 6XC2, 6XC4, 7JMP, 7JMO, 6XKQ, and 6XKP) on the three-dimensional structure of six neutralizing antibodies (CC12.1, CC12.3, COVA2-39, COVA2-04, CV07-250, and CV07-270) that bind to the spike glycoprotein of SARS-CoV-2 was used in these studies (6) . Using the Spanner program, we predicted the three-dimensional structure of the SARS-CoV-2 spike glycoprotein Y453F mutant based on PDB (ENTITY SEQ 6VW1_1). We investigated the binding of the spike glycoprotein Y453F mutant of SARS-CoV-2 to human angiotensin-converting enzyme 2 (ACE2) and determined the affinity of the spike glycoprotein Y453F mutant of SARS-CoV-2 to six neutralizing monoclonal antibodies using the MOE program (three-dimensional protein structure modeling, protein docking analysis: MOLSIS Inc., Tokyo, Japan) and Cn3D macromolecular structure viewer. The Y453F mutation did not affect the three-dimensional structure of conventional SARS-CoV-2 spike glycoproteins. From the present study, it was clarified that the binding property of the spike glycoprotein Y453F mutant to human ACE2 was slightly weaker than that of the conventional SARS-CoV-2 spike glycoprotein ( Figure 1A ). This was due to the replacement of Tyr at position 453 by Phe, which was unable to form a hydrogen bond with His at position 34 in human ACE2. The present study revealed that the affinity between the spike glycoprotein Y453F mutant and four of the six monoclonal antibodies (CC12.1, CC12.3, COVA2-39, COVA2-04, CV07-250, CV07-270) examined was clearly weak compared with the conventional SARS-CoV-2 spike glycoprotein ( Figure 1B , Table 1 ). It is considered that the affinity between the appropriate amino acid residues in the variable region of the antibody and the spike glycoprotein Y453F mutant was diminished owing to weak recognition of the monoclonal antibody to spike glycoproteins. To the best of our knowledge, data on all SARS-CoV-2 mutants have not been published till date. Therefore, it is unclear whether SARS-CoV-2 mutants detected in people working on mink farms are actually derived from the farmed minks. However, in the present study the subspecies of SARS-CoV-2 derived from farmed minks has been observed in the group of infected people, and the virus mutants that were inherited by infected individuals. Mutations in SARS-CoV-2 that lead to generation of SARS-CoV-2 subspecies, have made humans and animals susceptible to infection through easy propagation in the host, thereby making it difficult to identify the effects of therapeutic agents or vaccines for COVID-19. Moreover, the spread of SARS-CoV-2 variants mediated by millions of infected farmed mink is uncontrolled; consequently, raising a concern that infection of SARS-CoV-2 mutants that cause serious symptoms in humans may spread globally. Data are available on various websites and have also been made publicly available (more information can be found in the first paragraph of the Results section). The authors declare no potential conflicts of interest. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The interaction between Angiotensin-converting enzyme 2 (ACE2) (green) and residues of the conventional receptor binding domain (RBD) or RBD Y453F mutant (purple) is shown using the three-dimensional structure model. It is speculated that the Y453 amino acid residue of the conventional RBD is hydrogen-bonded to the H34 amino acid residue of human ACE2. However, the binding between the F453 amino acid residue of the RBD mutant and the H34 amino acid residue of human ACE2 is presumed to be slightly weak. From these results, the affinity between the spike glycoprotein of the RBD Y453F mutant and human ACE2 is presumed to be slightly weak compared with the conventional RBD. (B) The interaction between the heavy chain (purple) and light chain (brown) of neutralizing monoclonal antibody CC12.1 and residues of the conventional RBD or RBD Y453F mutant (green) is shown using the three-dimensional structure model. It is speculated that the Y453 amino acid residue of the conventional RBD is hydrogen-bonded to the D92 amino acid residue of the light chain and D97 amino acid residue of the heavy chain of neutralizing monoclonal antibody CC12.1. However, the binding between the F453 amino acid residue of the RBD mutant and the D92 and D97 amino acid residues of neutralizing monoclonal antibody CC12.1 is presumed to be weak. From these results, the affinity between the spike glycoprotein of the RBD Y453F mutant and neutralizing monoclonal antibody CC12.1 is presumed to be low compared with the conventional RBD. The three-dimensional structure models are shown by Cn3D macromolecular structure viewer. The binding of the spike glycoprotein Y453F mutant of SARS-CoV-2 to human ACE2 and the affinity of the spike glycoprotein Y453F mutant of SARS-CoV-2 to six neutralizing monoclonal antibodies were investigated using the MOE program (three-dimensional protein structure modeling, protein docking analysis: MOLSIS Inc., Tokyo, Japan) and Cn3D macromolecular structure viewer. Binding energy calculated by the MOE program is shown in the table. Coronavirus rips through Dutch mink farms, triggering culls Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans Subspecies of SARS-CoV-2 from farmed minks might be a threat to humans COVID mink analysis shows mutations are not dangerous-yet Structural basis of receptor recognition by SARS-CoV-2 Structural basis of a shared antibody response to SARS-CoV-2 We thank Professor Richard A. Young (Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA) for his research assistance. This study was supported in part by grants from the Japan Ministry of Education, Culture, Science and Technology (No. The transparency document associated with this article can be found in the online version at http://.