Single-Domain Antibodies Efficiently Neutralize SARS-CoV-2 Variants of Concern

Abstract

Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma, Delta and Omicron variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern.

Keywords: COVID-19; SARS-CoV-2; VHH; VOC; nanobodies; neutralizing antibodies; single-domain antibodies.

Figure 1

Isolation of RBD-specific nanobodies. (A) Immunization schedule. Bactrian camel was immunized with 100 μg RBD subcutaneously (with complete Freund`s adjuvant), followed by four consecutive immunization with 100 μg RBD subcutaneously (with incomplete Freund`s adjuvant). Blood samples were collected before immunization and five days after the last immunization. (B) RBD-specific antibodies in camel serum before and after immunization, detected by ELISA. The assay reveals a strong positive RBD-specific serological activity 5 days after the last immunization. (C) ELISA-based RBD-binders screening. A total of 212 individual clones with a strong positive ELISA signal were selected for sequencing. (D) Phylogenetic tree showing sequence diversity of 39 unique VHH clones from this study and four previously described single-domain antibodies of C. bactrianus (20), blue squares –previously described single-domain antibodies of C. bactrianus, green squares – the clones selected for further analysis.

Figure 2

Characterization of the selected nanobodies. (A) RBD-binding activity of nanobodies by ELISA and neutralization activity of nanobodies by microneutralization assay using live SARS-CoV-2 virus. The minimal neutralizing concentration was defined as the lowest antibody concentration (highest antibody dilution) that completely inhibited the cytopathic effect of the virus in two or three from the three replicable wells. (B) Kinetic parameters of nanobodies interaction with RBD by SPR. Association (k_on), dissociation (k_off), maximal analyte binding capacity (R_max), equilibrium association constants (K_A), equilibrium dissociation constants (K_D) and Chi² for VHHs binding to RBD. (C) SPR-based epitope binning experiments, in-tandem format. The first saturating antibody indicated on the top row, the second competing antibody indicated on the left column. (D) Blocking of ACE2-RBD interaction measured by competitive ELISA.