Spike protein antibodies mediate the apparent correlation between SARS-CoV-2 nucleocapsid antibodies and neutralization test results (preprint)

ObjectivesSARS-CoV-2 infection induces the formation of different antibodies. However, not all of which might prevent the virus from entering the cell, although their concentrations correlate with the titers of viral neutralization tests (NTs). Antibodies against the viral nucleocapsid (NC), e.g., can be classified as such. We aimed to prove the hypothesis that the apparent correlation between NC-antibody levels and NT-titers is mediated by simultaneously occurring antibodies against viral spike-protein components. MethodsWe included 64 individuals with previous SARS-CoV-2 infection (>14d after symptom onset). SARS-CoV-2 antibodies against the NC (Roche total antibody ECLIA, Abbott IgG CMIA) and spike-protein (Technozym RBD ELISA, DiaSorin S1/S2 CLIA) were measured, and neutralization tests were performed. The effect of spike-protein antibodies on the correlation between NC-antibodies and NT-titers was evaluated by partial correlation and mediation analyses. ResultsBoth tested assays assessing antibodies against the NC correlated significantly with NT titers: Abbott {rho}=0.742, P<0.0001; Roche {rho}=0.365, P<0.01. However, when controlling the rank correlations for the presence of RBD or S1/S2 antibodies, correlation coefficients dropped to {rho}=0.318/{rho}=0.329 (P<0.05/P<0.01), respectively for Abbott and vanished for Roche. As a result, only a maximum of 11% of NT titer variability could be explained by NC-antibody levels. ConclusionsOur data suggest that the apparent correlation between NC antibodies and NT titers is strongly mediated by co-occurring RBD antibody concentrations. To avoid falsely implied causal relationships, all correlation analyses of non-spike-associated antibody assays and neutralization assays should include a partial correlation analysis to exclude a possible mediator effect of spike-associated antibodies.

Antibody Seroprevalence and Rate of Asymptomatic Infections With SARS-CoV-2 in Austrian Hospital Personnel (preprint)

Context On March 11, the World Health Organization (WHO) announced the current corona virus disease 2019 (COVID-19) outbreak as a pandemic. The first laboratory-confirmed case of COVID-19 in Austria was announced on February 27, 2020. Since then, the incidence of infection followed an exponential increase until a complete lockdown in March 2020. Thereafter easing of restrictions was gradually introduced and until mid-August daily infections remained mostly below 5 per 100.000 population. Objectives: The aims of this study are to determine i) how many employees in Austrian trauma hospitals and rehabilitation facilities have virus specific IgG and IgM, and/or neutralizing antibodies against SARS-CoV-2, ii) how many are active virus carriers (symptomatic and asymptomatic) during the study, iii) the antibody decline in seropositive subjects over a period of around six months, and iv) the utility of rapid antibody tests for outpatient screening. Study Design Open uncontrolled observational cross-sectional study. Setting: /Participants A total of 3301 employees in 11 Austrian trauma hospitals and rehabilitation facilities of the Austrian Social Insurance for Occupational Risks (AUVA) participated in the study. Study Interventions and Measures Rapid antibody tests for SARS-CoV-2 specific IgG and IgM antibodies, and RT-PCR tests based on oropharyngeal swab samples, as well as laboratory-based antibody tests using ELISA/PRNT were performed. The tests were conducted twice, with an interval of 42.4±7.7 (Min=30, Max=64) days. Additionally, participants filled out a questionnaire including questions related to personal health, traveling activities, living situation, as well as inquiries of symptoms and comorbidities. Antibody positive tested participants were re-tested with ELISA/PRNT tests at a third time point on average 188.0±12.8 days after their initial test. Results: In our study cohort, only 27 out of 3301 participants (0.81%) had a positive antibody test at any time point during the study confirmed via neutralization test. Among participants who had positive test results in either of the antibody tests, 50.4% did not report any symptoms consistent with common manifestations of COVID-19 during the study period or within the preceding six weeks. In the group who tested positive during or prior to study inclusion the most common symptoms of an acute viral illness were rhinitis (21.9%), and loss of taste and olfactory sense (21.9%).The rapid antibody test was generally more sensitive based on serum (sensitivity=86.6%) as compared to whole blood (sensitivity=65.4). Concerning both ELISA tests overall the Roche test detected 24 (sensitivity=88.9%) and the Diasorin test 22 positive participants (sensitivity=81.5%).In participants with a positive PRNT, a significant decrease in PRNT concentration from 31.8±22.9 (Md=32.0) at T1 to 26.1±17.6 (Md=21.3) at T2 to 21.4±13.4 (Md=16.0) at T3 (c 2 =23.848, df=2, p<0.001) was observed (c 2 =23.848, df=2, p<0.001) – with an average time of 42.4±7.7 days between T1 and T2 and 146.9±13.8 days between T2 and T3. Conclusions: During the study period (May 11 th – December 21 th ) only 0.81% were tested positive for antibodies in our study cohort. The antibody concentration decreases significantly over time with 14.8% (4 out of 27) losing detectable antibodies.

Clinical validation of the quantitative Siemens SARS-CoV-2 spike IgG assay (sCOVG) reveals improved sensitivity and a good correlation with virus neutralization titers (preprint)

ObjectivesSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections cause Coronavirus Disease 2019 (COVID-19) and induce a specific antibody response. Serological assays detecting IgG against the receptor binding domain (RBD) of the spike (S) protein are useful to monitor the immune response after infection or vaccination. The objective of our study was to evaluate the clinical performance of the Siemens SARS-CoV-2 IgG (sCOVG) assay. MethodsSensitivity and specificity of the Siemens sCOVG test were evaluated on 178 patients with SARS-CoV-2-infection and 160 pre-pandemic samples in comparison with its predecessor test COV2G. Furthermore, correlation with virus neutralization titers was investigated on 134 samples of convalescent COVID-19 patients. ResultsSpecificity of the sCOVG test was 99.4% and sensitivity was 90.5% (COV2G assay 78.7%; p<0.0001). S1-RBD antibody levels showed a good correlation with virus neutralization titers (r=0.843; p<0.0001) and an overall qualitative agreement of 98.5%. Finally, median S1-RBD IgG levels increase with age and were significantly higher in hospitalized COVID-19 patients (median levels general ward: 25.7 U/ml; intensive care: 59.5 U/ml) than in outpatients (3.8 U/ml; p<0.0001). ConclusionsPerformance characteristics of the sCOVG assay have been improved compared to the predecessor test COV2G. Quantitative SARS-CoV-2 S1-RBD IgG levels could be used as a surrogate for virus neutralization capacity. Further harmonization of antibody quantification might assist to monitor the humoral immune response after COVID-19 disease or vaccination.

Antibody seroprevalence and rate of asymptomatic infections with SARS-CoV-2 in Austrian hospital personnel. (preprint)

Context: On March 11, the World Health Organization (WHO) announced the current corona virus disease 2019 (COVID-19) outbreak as a pandemic. The first laboratory-confirmed case of COVID-19 in Austria was announced on February 27, 2020. Since then, the incidence of infection followed an exponential increase until a complete lockdown in March 2020. Thereafter easing of restrictions was gradually introduced and until mid-August daily infections remained mostly below 5 per 100.000 population. Objectives: The aims of this study are to determine i) how many employees in Austrian trauma hospitals and rehabilitation facilities have virus specific IgG and IgM, and/or neutralizing antibodies against SARS-CoV-2, ii) how many are active virus carriers (symptomatic and asymptomatic) during the study, iii) the antibody decline in seropositive subjects over a period of around six months, and iv) the utility of rapid antibody tests for outpatient screening. Study Design: Open uncontrolled observational cross-sectional study. Setting/Participants: A total of 3301 employees in 11 Austrian trauma hospitals and rehabilitation facilities of the Austrian Social Insurance for Occupational Risks (AUVA) participated in the study. Study Interventions and Measures: Rapid antibody tests for SARS-CoV-2 specific IgG and IgM antibodies, and RT-PCR tests based on oropharyngeal swab samples, as well as laboratory-based antibody tests using ELISA/PRNT were performed. The tests were conducted twice, with an interval of 42.4{+/-}7.7 (Min=30, Max=64) days. Additionally, participants filled out a questionnaire including questions related to personal health, traveling activities, living situation, as well as inquiries of symptoms and comorbidities. Antibody positive tested participants were re-tested with ELISA/PRNT tests at a third time point on average 188.0{+/-}12.8 days after their initial test. Results: In our study cohort, only 27 out of 3301 participants (0.81%) had a positive antibody test at any time point during the study confirmed via neutralization test. Among participants who had positive test results in either of the antibody tests, 50.4% did not report any symptoms consistent with common manifestations of COVID-19 during the study period or within the preceding six weeks. In the group who tested positive during or prior to study inclusion the most common symptoms of an acute viral illness were rhinitis (21.9%), and loss of taste and olfactory sense (21.9%). The rapid antibody test was generally more sensitive based on serum (sensitivity=86.6%) as compared to whole blood (sensitivity=65.4). Concerning both ELISA tests overall the Roche test detected 24 (sensitivity=88.9%) and the Diasorin test 22 positive participants (sensitivity=81.5%). In participants with a positive PRNT, a significant decrease in PRNT concentration from 31.8{+/-}22.9 (Md=32.0) at T1 to 26.1{+/-}17.6 (Md=21.3) at T2 to 21.4{+/-}13.4 (Md=16.0) at T3 (chi-squared=23.848, df=2, p<0.001) was observed (chi-squared=23.848, df=2, p<0.001) - with an average time of 42.4{+/-}7.7 days between T1 and T2 and 146.9{+/-}13.8 days between T2 and T3. Conclusions: During the study period (May 11th - December 21th) only 0.81% were tested positive for antibodies in our study cohort. The antibody concentration decreases significantly over time with 14.8% (4 out of 27) losing detectable antibodies.

A comprehensive antigen production and characterization study for easy-to-implement, highly specific and quantitative SARS-CoV-2 antibody assays (preprint)

Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection. While first-generation antibody tests have primarily provided qualitative results with low specificity, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Here, we describe two quantitative ELISA antibody tests based on the SARS-CoV-2 spike receptor-binding domain and the nucleocapsid protein. Comparative expression in bacterial, insect, mammalian and plant-based platforms enabled the identification of new antigen designs with superior quality and high suitability as diagnostic reagents. Both tests scored excellently in clinical validations with multi-centric specificity and sensitivity cohorts and showed unprecedented correlation with SARS-CoV-2 neutralization titers. Orthogonal testing increased assay specificity to 99.8%, thereby enabling robust serodiagnosis in low-prevalence settings. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19.

Covid-19 serology in nephrology health care workers (preprint)

Background: Chronic kidney disease patients show a high mortality in case of a SARS-CoV-2 infection. Thus, to be informed on Nephrology personnel's sero-status might be crucial for patient protection. However, limited information exists about the presence of SARS-CoV-2 antibodies in asymptomatic individuals. Methods: We examined the seroprevalence of SARS-CoV-2 IgG and IgM antibodies among health care workers of a tertiary care kidney center during the peak phase of the Covid-19 crisis in Austria using an orthogonal test strategy and a total of 12 commercial nucleocapsid protein or spike glycoprotein based assays as well as Western blotting and a neutralization assay. Results: At baseline 60 of 235 study participants (25.5%, 95% CI: 20.4-31.5) were judged to be borderline positive or positive for IgM or IgG using a high sensitivity/low specificity threshold in one test system. Follow-up analysis after about two weeks revealed IgG positivity in 12 (5.1%, 95% CI: 2.9-8.8) and IgM positivity in six (2.6%, 95% CI: 1.1-5.6) in at least one assay. 2.1% (95% CI: 0.8-5.0) of health care workers showed IgG nucleocapsid antibodies in at least two assays. By contrast, positive controls with proven Covid-19 showed antibody positivity among almost all test systems. Moreover, serum samples obtained from health care workers did not show SARS-CoV-2 neutralizing capacity, in contrast to positive controls. Conclusions: Using a broad spectrum of antibody tests the present study revealed inconsistent results for SARS-CoV-2 seroprevalence among asymptomatic individuals, while this was not the case among Covid-19 patients.