Assessment of Immunogenic and Antigenic Properties of Recombinant Nucleocapsid Proteins of Five SARS-CoV-2 Variants in a Mouse Model

Figure 1. Assessment of homologous anti-N antibody levels in serum samples of mice immunized with B.1 (blue), B.1.351(brown), P.1 (green), B.1.617.2 (magenta) and B.1.1.529 (red). Hereafter, the color designations of the N proteins of different SARS-CoV-2 VOCs are the same. The OD450 values are shown in (a), and the area under the OD450 curve (AUC) values are shown in (b). These data were compared using ANOVA with the Tukey post hoc test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 2. N-based ELISAs of serum samples of mice immunized with B.1 (A, blue), B.1.351 (B, brown), P.1 (C, green), B.1.617.2 (D, magenta) and B.1.1.529 (E, red). The left panel shows the mean OD450 values in ELISAs with the indicated N antigen. The area under the OD450 curve (AUC) values for each serum sample tested against five N antigens are shown in the middle panel. The right panel shows the AUC values normalized to the homologous antigen. These data were compared using the Wilcoxon matched-pairs test. * p < 0.05, ** p < 0.01.

Figure 3. Scheme of N-protein domains and variable amino acid residues characterizing five different variants of the SARS-CoV-2 virus used in this work. The locations of the functional domains of the N protein are given according to [9]. The substitutions present in the N-protein sequences of the B.1.351 (brown), P.1 (green), B.1.617.2 (magenta) and B.1.1.529 (red) strains are indicated with arrows.

Figure 4. SDS-PAGE (a) and Western blot (b) analyses of the purified anti-N antibodies. The upper panel of the SDS-PAGE shows the results of the analysis in nonreducing conditions, and the lower panel shows the results of the analysis in the presence of 1% β-mercaptoethanol.

Figure 5. Analysis of specificity of N-specific mAbs in a Vero E6 cell model. (a) Dependence of absorbance intensity (OD450) on the concentration (µg/mL) of anti-N mAbs or 14C2 mAbs (NC, anti-influenza M2e antibodies). Vero E6 cells were infected with the B.1, B.1.617.2 or B.1.1.529 SARS-CoV-2 variant, followed by cell ELISA with indicated mAbs. (b) Immunocytochemical analysis of SARS-CoV-2 N protein in infected Vero E6 cells after 48 h of incubation. The different patterns of cytopathic effects caused by three different strains of SARS-CoV-2 are visualized. The positive red staining was developed as a result of treatment with the NCL2 and NCL10 (except for the B.1.1.529 detection) mAbs. The mock comprised the untreated cells. Scale bars: 100 µm.