Monoclonal Antibodies to S and N SARS-CoV-2 Proteins as Probes to Assess Structural and Antigenic Properties of Coronaviruses

Abstract

Antibodies targeting the spike (S) and nucleocapsid (N) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential tools. In addition to important roles in the treatment and diagnosis of infection, the availability of high-quality specific antibodies for the S and N proteins is essential to facilitate basic research of virus replication and in the characterization of mutations responsible for variants of concern. We have developed panels of mouse and rabbit monoclonal antibodies (mAbs) to the SARS-CoV-2 spike receptor-binding domain (S-RBD) and N protein for functional and antigenic analyses. The mAbs to the S-RBD were tested for neutralization of native SARS-CoV-2, with several exhibiting neutralizing activity. The panels of mAbs to the N protein were assessed for cross-reactivity with the SARS-CoV and Middle East respiratory syndrome (MERS)-CoV N proteins and could be subdivided into sets that showed unique specificity for SARS-CoV-2 N protein, cross-reactivity between SARS-CoV-2 and SARS-CoV N proteins only, or cross-reactivity to all three coronavirus N proteins tested. Partial mapping of N-reactive mAbs were conducted using truncated fragments of the SARS-CoV-2 N protein and revealed near complete coverage of the N protein. Collectively, these sets of mouse and rabbit monoclonal antibodies can be used to examine structure/function studies for N proteins and to define the surface location of virus neutralizing epitopes on the RBD of the S protein.

Keywords: COVID-19; SARS-CoV-2; antibodies; coronavirus; nucleocapsid protein; spike protein.

Figure 1

(a) Cell lysates and supernatants were collected from 293 cells transfected with constructs expressing S (+S) or N (–S) proteins of SARS-CoV-2 and subjected to Western blot analysis. Blots were probed with rabbit monoclonal #14 or actin (loading control for cell lysate only). (b) 293T cells transfected with a plasmid expressing SARS-CoV-2 S protein and subjected to IF analysis with S8.C mouse mAb or S #14 rabbit mAb at 24 h post-transfection.

Figure 2

(a) Western blot showing purified N proteins from SARS-CoV, SARS-CoV-2, and MERS-CoV probed with monoclonal N antibodies 9.10B, 8.5H, and 10.29B. A Coomassie-stained polyacrylamide gel shows the equal loading of the proteins. (b) 293T cells transfected with a plasmid expressing SARS-CoV-2 or the SARS-CoV N protein were fixed at 24 h post-transfection and probed with the rabbit polyclonal serum, or 7.2A, 8.5H, and 10.29B mouse monoclonal antibodies.

Figure 3

Schematic depicting the regions of the N protein targeted by each antibody based on competitive ELISA results.