Homologous and heterologous serological response to the N-terminal domain of SARS-CoV-2 in humans and mice

Abstract

The increasing numbers of infected cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses serious threats to public health and the global economy. Most SARS-CoV-2 neutralizing antibodies target the receptor binding domain (RBD) and some the N-terminal domain (NTD) of the spike protein, which is the major antigen of SARS-CoV-2. While the antibody response to RBD has been extensively characterized, the antigenicity and immunogenicity of the NTD protein are less well studied. Using 227 plasma samples from COVID-19 patients, we showed that SARS-CoV-2 NTD-specific antibodies could be induced during infection. As compared to the results of SARS-CoV-2 RBD, the serological response of SARS-CoV-2 NTD is less cross-reactive with SARS-CoV, a pandemic strain that was identified in 2003. Furthermore, neutralizing antibodies are rarely elicited in a mice model when NTD is used as an immunogen. We subsequently demonstrate that NTD has an altered antigenicity when expressed alone. Overall, our results suggest that while NTD offers a supplementary strategy for serology testing, it may not be suitable as an immunogen for vaccine development.

Keywords: COVID-19; N-terminal domain; NTD; SARS-CoV-2; immunogen; serology.

Figure 1

Patient serological responses to SARS‐CoV‐2 NTD and RBD protein (A and B) Binding of plasma from SARS‐CoV‐2 infected patients to SARS‐CoV‐2 NTD protein (A) and RBD protein (B) were measured during the days symptom after onset by ELISA assay. The mean OD₄₅₀ ELISA binding values calculated after testing each plasma sample in duplicate are shown. The plamsa sample from healthy donors were used as negative control. The ELISA cutoff value of NTD and RBD protein were 0.3272 and 0.2607, respectively (mean + three standard deviations). (C) Pearson correlation (r) was used to assess the relationship between measured SARS‐CoV‐2 serological binding responses to SARS‐CoV‐2 RBD and NTD protein in the SARS‐CoV‐2 infected patients at consequent time periods. Each sample was tested as duplicates in each assay and the results were confirmed by two independent experiments.

Figure 2

Cross‐reactive serological response to NTD and RBD protein between SARS‐CoV and SARS‐CoV‐2 (A and B) Pearson correlation (r) was used to evaluate the binding capacity of plasma to SARS‐CoV and SARS‐CoV‐2 NTD (A) and RBD (B) protein from 227 SARS‐CoV‐2 infected patients. The ELISA cutoff value of NTD protein to SARS‐CoV and SARS‐CoV‐2 were 0.5939 and 0.3272, and RBD protein to SARS‐CoV and SARS‐CoV‐2 were 0.2867 and 0.2607, respectively (mean + three standard deviations). Each sample was tested as duplicates in each assay and the results were confirmed by two independent experiments.

Figure 3

Cross‐reactive serological response to human coronaviruses between COVID‐19 patients and healthy donors (A and B) Binding of plasma samples to SARS‐CoV‐2 NTD (A), SARS‐CoV‐2 RBD (B), 229E‐Spike (C), NL63‐Spike (D), HKU1‐Spike (E), and OC43‐Spike protein (F) were tested by ELISA assay from 118 COVID‐2019 patients and age‐ and sex‐matched healthy donors. The OD₄₅₀ value from each dot in the figure was taken by means of two replicates in the same experiment. p‐Values were caluated using two‐tailed paired t‐test (***p < 0.001). Error bars repeesent strandard deviation. Each sample was tested as duplicates in each assay and the results were confirmed by two independent experiments.

Figure 4

Serological binding and neutralizing capacity against SARS‐CoV and SARS‐CoV‐2 by NTD protein immunization (A) Binding of plasma from SARS‐CoV‐2 NTD protein immunized mice, SARS‐CoV NTD protein immunized mice, live SARS‐CoV‐2 immunized mice and live SARS‐CoV immunized mice against SARS‐CoV and SARS‐CoV‐2 NTD protein were measured by ELISA assay. The mean OD₄₅₀ values calculated after detecting each plasma sample in duplicate are shown. (B) Neutralization activities of plasma from mice immunized with SARS‐CoV‐2 NTD protein, SARS‐CoV NTD protein, live SARS‐CoV‐2 and live SARS‐CoV were measured. The value from each dot in the figure was tested by the means of two replicates in the same assay. (C) Binding of plasma from SARS‐CoV and SARS‐CoV‐2 NTD protein immunized mice against the full spike of SARS‐CoV‐2 or SARS‐CoV. (D) Binding of plasma from SARS‐CoV and SARS‐CoV‐2 NTD protein immunized mice against NL63‐Spike, 229E‐Spike, HKU1‐Spike and OC43‐Spike protein were tested by ELISA assay. The OD₄₅₀ value from each dot in the figure was taken by means of two replicates in the same experiment. p‐Values were caluated using two‐tailed t‐test. Error bars represent standard deviation. Each sample was tested as duplicates in each assay and the results were confirmed by two independent experiments.

Figure 5

Conservation of NTD protein surface residues between SARS‐CoV‐2 and SARS‐CoV. (A‐B) Surface residues of NTD (cyan) that are conserved between SARS‐CoV‐2 and SARS‐CoV are highlighted in orange on (A) the spike protein where two RBD are in the down conformation (pink) and one RBD is in the up conformation (purple), and on (B) NTD alone. (C) NTD antibody supersites [24] highlighted in blue. Oligomannoses (yellow) were modeled by GlyProt [50].