Seropositivity to Nucleoprotein to detect SARS-CoV-2 infections: a tool to detect breakthrough infections after COVID-19 vaccination (preprint)

BackgroundWith 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. MethodsMultiplex immunoassay data from 1,513 SARS-CoV-2 RT-qPCR-tested individuals (352 positive and 1,161 negative) with a primary infection and no 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). ResultsThe 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 naive participants. ConclusionsSerological 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.

Serological Evidence for Reinfection with SARS-CoV-2; An Observational Cohort Study (preprint)

Background: Cases of reinfection with SARS-CoV-2 are reported in increasing numbers. Since the required molecular proof of reinfection is challenging to obtain, we aimed to provide serological evidence of reinfection in a cohort of potential reinfection cases.Methods: The study comprises 38 RT-PCR confirmed reinfection cases, with a COVID-19 symptom-free interval of at least 8 weeks (range 57-133 days) since their first RT-PCR confirmed episode. Specific disease symptoms were retrieved from 22 cases by contact tracing and compared between the two disease episodes. The oropharyngeal specimens from 13 cases enabled adequate genomic sequence comparisons. Seventeen cases provided a serum specimen, of which 12 within 7 days after onset of symptoms. Antibody determinations included SARS-CoV-2-specific total Ig, IgM, IgG, avidity, and virus neutralization. Antibody data were compared to that of a control group of primary cases (n=86) in relation to time since onset of disease symptoms.Findings: Reinfection cases generally experienced fewer or milder symptoms. Five of 8 cases which passed genomic comparison between both disease episodes showed reinfection with a different lineage. From 12 reinfection cases that provided a serum sample within 7 days after onset of symptoms, 11/12 (92%) and 12/12 (100%) showed high levels of specific total Ig and IgG antibodies, respectively, compared to 1/23 (4%) and 2/23 (9%) within the control group. Virus neutralizing antibodies were detected in 9/12 (75%) reinfection cases, 5 of which were above a titer of 30. Serological discrimination diminished after 7 days, except for IgG avidity; all 17 reinfection cases had antibodies of higher avidity when compared to control cases.Interpretation: IgG concentration and avidity can be used as an additional diagnostic marker to confirm reinfection with SARS-Cov-2. Reinfection cases that show a rapid and effective secondary immune response are expected to clear the infection more effectively, thereby reducing contagiousness and clinical severity. Understanding this reinfection response is also important for breakthrough infections following vaccination.Funding Statement: This work was supported by the National Institute for Public Health and the Environment, The Netherlands.Declaration of Interests: None of the authors have an association that poses a conflict of interest.Ethics Approval Statement: Approved by the Medical-Ethical Review Committee of the University Medical Center Utrecht.