Human antibody recognition and neutralization mode on the NTD and RBD domains of SARS-CoV-2 spike protein

Figure 1. Two LCL supernatants suppressed CPE due to SARS-CoV-2 infection. (A) The number of cells with CPE in each panel of Fig. S1. (B) Two LCL supernatant (Clone ID: 9 and 13) were incubated with or without WT SARS-CoV-2 for 1 h. VeroE6-TMPRSS2 cells were infected with WT SARS-CoV-2 for 4 days in triplicate, which induced CPE. The number of cells with CPE was counted with ImageJ software. ****P < 0.001 (ANOVA).

Figure 2. Binding affinity of NIBIC-71 and 7G7 to the S protein. (A) The ability to bind to the S protein was evaluated with ELISA and serially diluted recombinant mAbs from 10 to 0.001 µg/mL in triplicate. Blue diamonds indicate NIBIC-71, green squares indicate 7G7/λ, red triangles indicate 7G7/κ, and cross markses indicate negative control mAb (N.C.). (B) SPR analyses for these mAbs. The SPR sensorgrams were obtained by injecting twofold serial dilutions of the S protein ranging from 80 to 0.625 nM for NIBIC-71, 7G7/λ, and 7G7/κ. Contact time and dissociation time were set as 240 s and 900 s, respectively. The kinetic values are shown in Table 2.

Figure 3. Neutralization mechanisms of NIBIC-71 and 7G7. (A) Truncated S proteins tagged with Flag and 6 × His were synthesized with the Bac-to-Bac baculovirus system. Purified proteins were dialyzed with PBS. (B) The binding regions of these mAbs were determined using ELISA with the recombinant proteins shown in A as antigens. The vertical axis shows normalized values (arbitrary unit) relative to absorbance obtained with ELISA against His. (C) and (D) Cryo-EM sharpened maps of the homotrimeric S protein complexed with NIBIC-71 (C) or 7G7/κ (D). The atomic model of S protein and the homology models of the Fabs are manually fitted into the maps. The regions corresponding to the two mAbs are shown in blue, green, and red. The heavy and light chains are shown in different colors. The NTD and RBD region in the S protein are shown in purple and yellow, respectively. One of S2’ and S1/S2 cleavage site are shown in red and blue circle, respectively. (E) The neutralizing activity of these mAbs was assessed with a luciferase assay in triplicate. VeroE6-TMRPSS2 cells were infected with WT SARS-CoV-2 introduced by nanoLuciferase. The cells were pre-incubated with or without serially diluted…

Figure 4. Analysis of in vivo neutralizing activity of NIBIC-71 and 7G7. Male 3-week-old Syrian hamsters that received 4 mg/kg of recombinant mAbs intravenously were infected with WT SARS-CoV-2 via the nasal route. (A) the schematic of in vivo infectious experiment. (B) Body weight (left) and rectal temperature (right) were monitored up to 4 days after infection (n = 3). Fold change is the value relative to before infection. (C) Viral load in the lung at 4 days after infection was shown as the means ± standard error of the mean for TCID50 (left) and copy number (right) (n = 3). (D) Histological analysis with IHC (N antigen) (upper) and HE staining (lower). SARS-CoV-2 was detected with an anti-nucleocapsid protein antibody. The brown staining indicated nucleocapsid (N antigens) of SARS-CoV-2. E. The proportion of the area with cellular infiltration was presented as mean ± standard error of the mean15. This value is the ratio of the HE-stained area to the whole area. These areas were obtained in 5 sections from each hamster. *P < 0.05; **P < 0.01, ****P < 0.001 (ANOVA). TCID50, tissue culture infectious dose.

Figure 5. Evaluation of in vitro neutralizing activity against the Delta and Omicron variants. (A) and (B) VeroE6-TMPRSS2 cells were infected with WT SARS-CoV-2, Delta, and Omicron with or without serially diluted NIBIC-71 (A) and 7G7/κ (B) from 10 to 0.001 µg/mL in quadruplicate. The number of wells with CPE was counted. Viral loads were presented as TCID50s. Inhibition efficiency (%) was calculated relative to values derived from the negative control samples without any mAbs. (C) and (D) Binding region of NIBIC-71 (C) and 7G7/κ (D). Homology models of the two Fabs are generated and manually fitted into the cryo-EM maps. Surfaces of the S proteins are shown in gray, and the mutation points in Delta B.1.617.2 (left panels) or Omicron BA.1 lineage (right panels) are shown in red.