Neutralizing and Enhancing Epitopes of the SARS-CoV-2 Receptor-Binding Domain (RBD) Identified by Nanobodies
Kanasap Kaewchim,
Kittirat Glab-ampai,
Kodchakorn Mahasongkram,
Thanatsaran Saenlom,
Watayagorn Thepsawat,
Monrat Chulanetra,
Kiattawee Choowongkomon,
Nitat Sookrung,
Wanpen Chaicumpa
Affiliations
Kanasap Kaewchim
Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Bangkok 10700, Thailand
Kittirat Glab-ampai
Center of Research Excellence in Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Bangkok 10700, Thailand
Kodchakorn Mahasongkram
Center of Research Excellence in Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Bangkok 10700, Thailand
Thanatsaran Saenlom
Center of Research Excellence in Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Bangkok 10700, Thailand
Watayagorn Thepsawat
Center of Research Excellence in Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Bangkok 10700, Thailand
Monrat Chulanetra
Center of Research Excellence in Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Bangkok 10700, Thailand
Kiattawee Choowongkomon
Department of Biochemistry, Faculty of Sciences, Kasetsart University, Bangkok 10900, Thailand
Nitat Sookrung
Center of Research Excellence in Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Bangkok 10700, Thailand
Wanpen Chaicumpa
Center of Research Excellence in Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Bangkok 10700, Thailand
Engineered nanobodies (VHs) to the SARS-CoV-2 receptor-binding domain (RBD) were generated using phage display technology. A recombinant Wuhan RBD served as bait in phage panning to fish out nanobody-displaying phages from a VH/VHH phage display library. Sixteen phage-infected E. coli clones produced nanobodies with 81.79–98.96% framework similarity to human antibodies; thus, they may be regarded as human nanobodies. Nanobodies of E. coli clones 114 and 278 neutralized SARS-CoV-2 infectivity in a dose-dependent manner; nanobodies of clones 103 and 105 enhanced the virus’s infectivity by increasing the cytopathic effect (CPE) in an infected Vero E6 monolayer. These four nanobodies also bound to recombinant Delta and Omicron RBDs and native SARS-CoV-2 spike proteins. The neutralizing VH114 epitope contains the previously reported VYAWN motif (Wuhan RBD residues 350–354). The linear epitope of neutralizing VH278 at Wuhan RBD 319RVQPTESIVRFPNITN334 is novel. In this study, for the first time, we report SARS-CoV-2 RBD-enhancing epitopes, i.e., a linear VH103 epitope at RBD residues 359NCVADVSVLYNSAPFFTFKCYG380, and the VH105 epitope, most likely conformational and formed by residues in three RBD regions that are spatially juxtaposed upon the protein folding. Data obtained in this way are useful for the rational design of subunit SARS-CoV-2 vaccines that should be devoid of enhancing epitopes. VH114 and VH278 should be tested further for clinical use against COVID-19.
