Tag-Free SARS-CoV-2 Receptor Binding Domain (RBD), but Not C-Terminal Tagged SARS-CoV-2 RBD, Induces a Rapid and Potent Neutralizing Antibody Response.

ABSTRACT

Recombinant proteins are essential in the development of subunit vaccines. In the design of many recombinant proteins, polyhistidine residues are added to the N- or C-termini of target sequences to facilitate purification. However, whether the addition of tag residues influences the immunogenicity of proteins remains unknown. In this study, the tag-free SARS-CoV-2 RBD and His-tag SARS-CoV-2 RBD proteins were investigated to determine whether there were any differences in their receptor binding affinity and immunogenicity. The results showed that the tag-free RBD protein had a higher affinity for binding with hACE2 receptors than His-tag RBD proteins (EC50 1.78 µM vs. 7.51 µM). On day 21 after primary immunization with the proteins, the serum ELISA titers of immunized mice were measured and found to be 11418 for those immunized with tag-free RBD and only 12.4 for His-tag RBD. Two weeks after the booster dose, tag-free-RBD-immunized mice demonstrated a significantly higher neutralizing titer of 1369 compared with 17.9 for His-tag-RBD-immunized mice. Furthermore, neutralizing antibodies induced by tag-free RBD persisted for up to 5 months and demonstrated greater cross-neutralization of the SARS-CoV-2 Delta variant. Evidence from Western blotting showed that the serum of His-tag-RBD-immunized mice recognized irrelevant His-tag proteins. Collectively, we conclude that the addition of a polyhistidine tag on a recombinant protein, when used as a COVID-19 vaccine antigen, may significantly impair protein immunogenicity against SARS-CoV-2. Antibody responses induced were clearly more rapid and robust for the tag-free SARS-CoV-2 RBD than the His-tag SARS-CoV-2 RBD. These findings provide important information for the design of antigens used in the development of COVID-19 subunit vaccines.