N-terminally truncated nucleocapsid protein of SARS-CoV-2

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) had led to a serious public health crisis, and no specific treatments or vaccines are available yet.

A nucleocapsid protein (NP)-based enzyme-linked immunosorbent assay (ELISA) detection method is not only important in disease diagnosis, but is required for the evaluation of vaccine efficacy during the development of an inactivated SARS-CoV-2 vaccine. In this study, we expressed both the NP and N-terminally truncated NP (ΔN-NP) of SARS-CoV-2 in an Escherichia coli expression system and described the purification of the soluble recombinant NP and ΔN-NP in details. The identities of the NP and ΔN-NP were confirmed with mass spectrometry.

We then used immunoglobulin G detection ELISAs to compare the sensitivity of NP and ΔN-NP in detecting anti-SARS-CoV-2 antibodies. ΔN-NP showed greater sensitivity than NP in the analysis of serially diluted sera from mice and rabbits vaccinated with inactive SARS-CoV-2 and in human sera diluted 1:400.

ΔN-NP showed a positive detection rate similar to that of the SARS-CoV-2 S protein in human sera. We conclude that ΔN-NP is a better serological marker than NP for evaluating the immunogenicity of inactivated SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten global public health. Several vaccines have already completed phase II clinical trials,1-4 although there are not appropriate therapies for COVID-19.5 Accurate serological diagnostic strategies are the first step in inhibiting the spread of SARS-CoV-2 and allow the qualitative and quantitative study of the immune responses to SARS-CoV-2.6, 7 The vaccine development process also requires that antibody responses and serological conversion can be determined accurately.

During the SARS-CoV outbreak in 2003, researchers considered the truncated nucleocapsid protein (NP) a good marker for diagnostic purposes.8 An enzyme-linked immunosorbent assay (ELISA) based on the NP of SARS-CoV-2 has also been used in the serodiagnosis of this novel COVID-19.9 An antibody directed against NP is more sensitive than one directed against the spike protein in detecting early infections.

10 However, there are some controversial findings of the nonspecific of NP. Guo et al.9 showed that recombinant whole NP did not cross-react with antibodies directed against other common coronaviruses. However, Yamaoka et al.11 reported that full-length (FL)-NP had a higher false-positive rate than N-terminally truncated NP (ΔN-NP) in sera diluted 1:100.

They concluded that ΔN-NP is better suited than FL-NP to the development of highly sensitive diagnostic assays for COVID-19. This suggests that an ΔN-NP-based ELISA has potential utility in evaluating the efficacy of inactivated SARS-CoV-2 vaccines. Because the antibody response to NP and its serological conversion in rodents and humans must be detectable during the development of inactivated SARS-CoV-2 vaccines, it is essential to enhance the sensitivity of such an assay to detect low levels of immunoglobulin G (IgG) in mice or humans, especially during the earlier period soon after vaccination.

To address this issue, we used ΔN-NP to develop ELISAs with which to detect anti-SARS-CoV-2 NP antibodies in mouse, rabbit, and human serum. Our results indicated that SARS-CoV-2 ΔN-NP is more sensitive than NP for evaluating the immune effects of an inactivated SARS-CoV-2 vaccine.