The Use of Heterologous Antigens for Biopanning Enables the Selection of Broadly Neutralizing Nanobodies Against SARS-CoV-2

Abstract

Background: Since the emergence of SARS-CoV-2 in the human population, the virus genome has undergone numerous mutations, enabling it to enhance transmissibility and evade acquired immunity. As a result of these mutations, most monoclonal neutralizing antibodies have lost their efficacy, as they are unable to neutralize new variants. Antibodies that neutralize a broad range of SARS-CoV-2 variants are of significant value in combating both current and potential future variants, making the identification and development of such antibodies an ongoing critical goal. This study discusses the strategy of using heterologous antigens in biopanning rounds. Methods: After four rounds of biopanning, nanobody variants were selected from a phage display library. Immunochemical methods were used to evaluate their specificity to the S protein of various SARS-CoV-2 variants, as well as to determine their competitive ability against ACE2. Viral neutralization activity was analyzed. A three-dimensional model of nanobody interaction with RBD was constructed. Results: Four nanobodies were obtained that specifically bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein and exhibit neutralizing activity against various SARS-CoV-2 strains. Conclusions: The study demonstrates that performing several rounds of biopanning with heterologous antigens allows the selection of nanobodies with a broad reactivity spectrum. However, the fourth round of biopanning does not lead to the identification of nanobodies with improved characteristics.

Keywords: AlphaFold; SARS-CoV-2; VHH; affinity selection; biopanning; molecular dynamics; nanobodies; phage library; protein–protein docking; recombinant antibodies; single-domain antibodies.

Figure 1

Phage display scheme showing antigens, eluate, and amplicon titres for each round.

Figure 2

Electrophoretic separation of synthesized nanobodies in 10% SDS-PAGE. Labels: M—molecular weight protein markers with the molecular weight in kDa indicated on the left (Precision Plus Protein™ Dual Xtra Prestained Protein Standards, Bio-Rad, Hercules, CA, USA); RC, SKP, KWL, PRV—purified recombinant nanobodies.

Figure 3

Binding of ACE2 to recombinant SARS-CoV-2 S protein trimers upon inhibition of interaction by nanobodies. The 100% interaction level is considered to be the signal of direct binding between the trimer and ACE2. Notations: PRV, KWL, SKP, RC—lysates of nanobody producers; E. coli (C−)—negative control producer, lysate of cells transformed with the pET21a(−) plasmid; VHH9 (C−)—nanobody specific to HIV-1, negative control of a heterologous nanobody; iB20—broad-neutralizing human monoclonal antibody against SARS-CoV-2 [32], positive control. One-way ANOVA showed statistically significant differences in the inhibition of ACE2 binding among nanobodies for each SARS-CoV-2 variant (Wuhan, Delta, and Omicron) with p < 0.0001.

Figure 4

Position of the KWL nanobody in the ACE2-binding domain. (a)—visualization of the ACE2–RBD complex (PDB ID 6VW1 [42]); (b)—statistically significant KWL–RBD–Wuhan complex; (c)—statistically significant KWL–RBD–Delta complex; (d)—statistically significant KWL–RBD–Omicron complex, obtained as a result of clustering the last 100 ns of MD simulation. For better visual perception, the structure of each protein, including α-helices and β-strands, is shown in different colors. In panel (a), ACE2 is shown in green, in panels (b–d), the KWL nanobody is shown in purple, RBD Wuhan in blue, RBD Delta in red, and RBD Omicron in green. Hydrogen bonds, salt bridges, and π-π stacking interactions are shown as yellow, purple, and blue dashed lines, respectively.

Figure 5

Loss of nanobody variant diversity during standard phage display procedures.