Seropositivity to Nucleoprotein to detect mild and asymptomatic SARS-CoV-2 infections: A complementary tool to detect breakthrough infections after COVID-19 vaccination?

Abstract

Background: With COVID-19 vaccine roll-out ongoing in many countries globally, monitoring of breakthrough infections is of great importance. Antibodies persist in the blood after a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Since COVID-19 vaccines induce immune response to the Spike protein of the virus, which is the main serosurveillance target to date, alternative targets should be explored to distinguish infection from vaccination.

Methods: Multiplex immunoassay data from 1,513 SARS-CoV-2 RT-qPCR-tested individuals (352 positive and 1,161 negative) without COVID-19 vaccination history were used to determine the accuracy of Nucleoprotein-specific immunoglobulin G (IgG) in detecting past SARS-CoV-2 infection. We also described Spike S1 and Nucleoprotein-specific IgG responses in 230 COVID-19 vaccinated individuals (Pfizer/BioNTech).

Results: The sensitivity of Nucleoprotein seropositivity was 85% (95% confidence interval: 80-90%) for mild COVID-19 in the first two months following symptom onset. Sensitivity was lower in asymptomatic individuals (67%, 50-81%). Participants who had experienced a SARS-CoV-2 infection up to 11 months preceding vaccination, as assessed by Spike S1 seropositivity or RT-qPCR, produced 2.7-fold higher median levels of IgG to Spike S1 ≥ 14 days after the first dose as compared to those unexposed to SARS-CoV-2 at ≥ 7 days after the second dose (p = 0.011). Nucleoprotein-specific IgG concentrations were not affected by vaccination in infection-naïve participants.

Conclusions: Serological responses to Nucleoprotein may prove helpful in identifying SARS-CoV-2 infections after vaccination. Furthermore, it can help interpret IgG to Spike S1 after COVID-19 vaccination as particularly high responses shortly after vaccination could be explained by prior exposure history.

Keywords: COVID-19; Multiplex immunoassay; Nucleoprotein; SARS-CoV-2; Serosurveillance; immunoglobulin G.

Fig. 1

Nucleoprotein and Spike S1 IgG responses to detect SARS-CoV-2 infections. In (A) IgG concentrations to Nucleoprotein (orange) and Spike S1 (green) are shown for four groups: RT-qPCR negative persons, RT-qPCR positive persons without symptoms, RT-qPCR positive persons with mild COVID-19 and RT-qPCR positive persons hospitalized with COVID-19, along with the threshold for seropositivity (dashed horizontal line). Data for 2 weeks to 6 months since infection are shown. In (B) specificity and sensitivity estimates with 95% confidence intervals are shown for Nucleoprotein (orange) and Spike S1 (green) seropositivity. Data for 2 weeks to 6 months since infection are shown. In (C) sensitivity estimates with 95% confidence intervals of Nucleoprotein (orange) and Spike S1 (green) seropositivity are shown over time for RT-qPCR positive persons with mild COVID-19; this does not include repeated samples from the same individuals. S1: Spike S1, N: Nucleoprotein, IgG: immunoglobulin G, BAU/ml: binding antibody units; NS: not significant (p > 0.05); *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Fig. 2

Nucleoprotein and Spike S1 IgG kinetics following COVID-19 vaccination. IgG measurements to Spike S1 (A) and Nucleoprotein (B) in infection-naïve individuals are shown over days since first vaccination. In (C) IgG measurements to Spike S1 are shown by prior exposure status and number of doses received, individuals were included if they were sampled ≥ 14 days after the first dose or ≥ 7 days after the second dose. In (A-C) the dashed horizontal line depicts the threshold for seropositivity. S1: Spike S1, N: Nucleoprotein, IgG: immunoglobulin G, BAU/ml: binding antibody units; NS: not significant (p > 0.05); *: p < 0.05; **: p < 0.01; ***: p < 0.001.