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Objectives Anti-nucleocapsid (NC) antibodies are produced in response to SARS-CoV-2 infection. Therefore, they are well suited for the detection of a previous infection. Especially in the case of seroprevalence studies or during the evaluation of a novel in-vitro diagnostic test, samples have been stored at Methods The preanalytical impact of short-term storage (84 [58–98] days) on Results After short-term storage, the observed changes did not exceed the extent that could be explained by analytical variability. In contrast, results after long-term storage were approximately 20% higher and seemed to increase with storage duration. This effect was independent of the biobank from which the samples were obtained. Accordingly, the sensitivity increased from 92.6 to 95.3% (p=0.008). However, comparisons with data from Anti-Spike protein assays, where these deviations were not apparent, suggest that this deviation could also be explained by the analytical variability of the qualitative Anti-NC assay. Conclusions Results from anti-NC antibodies are stable during short-term storage at

BackgroundAn understanding vaccine-dependent effects on protective and sustained humoral immune response is crucial to planning future vaccination strategies against coronavirus disease 2019 (COVID-19).MethodsIn this multicenter, population-based, cohort study including 4601 individuals after primary vaccination against COVID-19 ≥ 4 months earlier we compared factors associated with residual antibody levels against severe acute respiratory syndrome coronavirus-2 receptor-binding domain (RBD) across different vaccination strategies (BNT162b2, mRNA-1273, or ChAdOx1).ResultsOur main model including 3787 individuals (2 × BNT162b2, n = 2271; 2 × mRNA-1273, n = 251; 2 × ChAdOx1, n = 1265), predicted significantly lower levels of anti-RBD antibodies after 6 months in individuals vaccinated with ChAdOx1 (392.7 binding antibody units per milliliter [BAU/mL]) compared with those vaccinated with BNT162b2 (1179.5 BAU/mL) or mRNA-1273 (2098.2 BAU/mL). Vaccine-dependent association of antibody levels was found for age with a significant predicted difference in BAU/ml per year for BNT162b2 (−21.5; 95% confidence interval [CI], −24.7 to −18.3) and no significant association for mRNA-1273 (−4.0; 95% CI, −20.0 to 12.1) or ChAdOx1 (1.7; 95% CI, .2 to 3.1). The predicted decrease over time since full immunization was highest in mRNA-1273 (−23.4; 95% CI, −31.4 to −15.4) compared with BNT162b2 (−5.9; 95% CI, −7 to −4.8).ConclusionsOur study revealed population-based evidence of vaccine-dependent effects of age and time since full immunization on humoral immune response. Findings underline the importance of individualized vaccine selection, especially in elderly individuals.

Epstein-Barr virus (EBV) reactivation may be involved in long-COVID symptoms. Here we evaluated reactivation of parvovirus B19 and several viruses of the herpes family in patients with long-COVID syndrome, how vaccination affected viral interference, and how virus reactivation influenced clinical conditions. Clinical and laboratory data on 252 consecutive patients (97 vaccinated and 155 non-vaccinated) were recorded between April 2021–May 2022 (median 243 days post-COVID-19 infection). Viral IgG and IgM titers were compared between vaccinated or non-vaccinated patients, and age and sex-matched healthy controls. Vaccination was associated with significantly less frequent fatigue and multiorgan symptoms (P < 0.001), significantly less cumulative IgM positivity of the investigated viruses, significantly lower plasma levels of IgG subfractions 2 and 4, and significantly lower quantitative Cytomegalovirus (CMV) IgG, CMV IgM, and EBV IgM titers. These results indicate that anti-SARS-CoV2 vaccination interrupts viral crosstalk in patients with long-COVID syndrome. (ClinicalTrials.gov Identifier: NCT05398952)

To investigate the comparability of WHO standard referenced commercial SARS-CoV-2 antibody tests over three doses of BNT162b2 vaccine and up to 14 months.114 subjects (without previous SARS-CoV-2 infection or immunosuppressive medication) vaccinated with three doses of BNT162b2 were included in this study. Antibody levels were quantified 3 weeks after the first dose, 5-6 weeks and 7 months after the second dose, and 4-5 weeks and 4 months after the third dose using the Roche Elecsys SARS-CoV-2 S, the Abbott SARS-CoV-2 IgG II Quant, the DiaSorin LIAISON SARS-CoV-2 TrimericS IgG, the GenScript cPASS sVNT and the TECO sVNT assays.For each time point analyzed, systematic differences are evident between the results in BAU/mL of the three antibody binding assays. The assay ratios change in a time-dependent manner even beyond administering the third dose (Roche measuring 9 and 3 times higher than Abbott and DiaSorin, respectively). However, changes decrease in magnitude with increasing time intervals from the first dose. IgG-based assays show better agreement across them than with Roche (overall correlations: Abbott x DiaSorin: ρ = 0.94 vs. Abbott x Roche: ρ=0.89, p 0.0001; DiaSorin x Roche: ρ = 0.87, p 0.0001), but results are not interchangeable. The sVNTs suggest an underestimation of antibody levels by Roche and slight overestimation by both IgG assays after the first vaccine dose.Standardization of SARS-CoV-2 antibody binding assays still needs to be improved to allow reliable use of variable assay systems for longitudinal analyses.

Understanding vaccine-dependent effects on protective and sustained humoral immune response are crucial to plan further vaccination strategies against COVID-19. Population-based data comparing different vaccination strategies are lacking.This multicenter, population-based cohort study included 4,601 individuals ≥18 years of age after primary vaccination against COVID-19 at least four months ago (full immunization). We compared factors associated with residual antibody levels against SARS-CoV-2 receptor binding domain (RBD) across different vaccination strategies (BNT162b2, mRNA-1273, or ChAdOx1 nCoV-19 [ChAdOx1]).Our main model including 3,787 individuals (2xBNT162b2 n = 2,271, 2xmRNA-1273 n = 251, 2xChAdOx1 n = 1,265) predicted significantly lower levels of anti-RBD-antibodies after 6 months in individuals vaccinated with ChAdOx1 (392.7 BAU/ml) compared to those vaccinated with BNT162b2 (1179.5 BAU/ml) or mRNA-1273 (2098.2 BAU/ml). Vaccine-dependent association of antibody levels was found for age with a significant predicted difference in BAU/ml per year for BNT162b2 (-21.5 [95%CI -24.7 to -18.3]) and no significant association for mRNA-1273 (-4.0 [95%CI -20.0 to 12.1]) or ChAdOx1 (1.7 [95%CI 0.2 to 3.1]). The predicted decrease over time since full immunization was highest in mRNA-1273 (-23.4 [95%CI -31.4 to -15.4]) compared to BNT162b2 -5.9 [95%CI -7 to -4.8]). Higher antibody levels were observed for individuals with systemic adverse events upon vaccination and current smoking (BNT162b2), for days between first and second vaccination (BNT162b2 and ChAdOx1) and for absence of comorbidities (all).Our study revealed population-based evidence of vaccine-dependent effects of age and time since full immunization on humoral immune response. Findings underline the importance of an individualized vaccine selection, especially in elderly individuals.

ObjectivesVarious commercial anti-Spike SARS-CoV-2 antibody tests are used for studies and in clinical settings after vaccination. An international standard for SARS-CoV-2 antibodies has been established to achieve comparability of such tests, allowing conversions to BAU/ml. This study aimed to investigate the comparability of antibody tests regarding the timing of blood collection after vaccination.MethodsFor this prospective observational study, antibody levels of 50 participants with homologous AZD1222 vaccination were evaluated at 3 and 11 weeks after the first dose and 3 weeks after the second dose using two commercial anti-Spike binding antibody assays (Roche and Abbott) and a surrogate neutralization assay.ResultsThe correlation between Roche and Abbott changed significantly depending on the time point studied. Although 3 weeks after the first dose, Abbott provided values three times higher than Roche, 11 weeks after the first dose, the values for Roche were twice as high as for Abbott, and 3 weeks after the second dose even 5-6 times higher.ConclusionsThe comparability of quantitative anti-Spike SARS-CoV-2 antibody tests is highly dependent on the timing of blood collection after vaccination. Therefore, standardization of the timing of blood collection might be necessary for the comparability of different quantitative SARS-COV-2 antibody assays.Abstract Figure

ObjectivesOur objective was to determine whether SARS-CoV-2 antibody levels after the first dose can predict the final antibody response and whether this is dependent on the vaccine type.Methods69 BNT162b2 (Pfizer/BioNTech) and 55 AZD1222 (AstraZeneca) vaccinees without previous infection or immunosuppressive medication were included. Anti-body levels were quantified 3 weeks after dose 1, in case of AZD1222 directly before boostering (11 weeks after dose 1) and 3 weeks after dose 2, with the Roche SARS-CoV-2 S total antibody assay.ResultsPre-booster (BNT162b2: 80.6 [25.5-167.0] BAU/mL, AZD1222: 56.4 [36.4-104.8] BAU/mL, not significant) and post-booster levels (BNT162b2: 2,092.0 [1,216.3-4,431.8] BAU/mL, AZD1222: 957.0 [684.5-1,684.8] BAU/mL, pConclusionsIn conclusion, our data suggest that antibody levels quantified by the Roche Elecsys SARS-CoV-2 S assay before the booster shot could infer post-booster responses to BNT162b2, but not to AZ1222. In addition, we found a vaccine-dependent effect on antibody responses, suggesting a possible link between vaccine response and vector immunity.

BackgroundSerological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems are often poor, leaving room for false-positive and false-negative results. However, conventional methods were used to increase specificity and decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the ‘sensitivity improved two-test’ or ‘SIT²’ algorithm.MethodsSIT² involves confirmatory retesting of samples with results falling in a predefined retesting zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT² to single tests and orthogonal testing (OTA) in an Austrian cohort (1117 negative, 64 post-COVID-positive samples) and validated the algorithm in an independent British cohort (976 negatives and 536 positives).ResultsThe specificity of SIT² was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT² when compared with single tests or OTA. SIT² allowed correct identification of infected individuals even when a live virus neutralisation assay could not detect antibodies. Compared with single testing or OTA, SIT² significantly reduced total test errors to 0.46% (0.24–0.65) or 1.60% (0.94–2.38) at both 5% or 20% seroprevalence.ConclusionFor SARS-CoV-2 serology, SIT² proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases.

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 ρ=0.742, PConclusionsOur 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.

Background There is preliminary evidence that individuals with previous SARS‐CoV‐2 infections exhibit a more pronounced antibody response. However, these assumptions have not yet been supported by data obtained through various CE‐marked tests. This study aimed to close this gap. Methods Sixty‐nine seronegatives and 12 individuals post‐SARS‐CoV‐2 infection (tested by CE‐labelled Roche NC immunoassay or PCR‐confirmed assay) were included 21 ± 1 days after receiving the first dose of the Pfizer/BioNTech BNT162b2 vaccine. Antibody response to viral spike protein (S) was assessed by CE‐labelled Roche S and DiaSorin S1/S2 assays and by a surrogate virus neutralization test (sVNT). Results After a single dose of BNT162b2, individuals after natural SARS‐CoV‐2 infection presented with markedly higher anti‐S levels than naïve individuals (Roche S: 9078.5 BAU/mL [5267.0‐24 298.5] vs 79.6 [24.7‐142.3]; and DiaSorin S1/S2: 1465.0 AU/mL [631.0‐5365.0] vs 63.7 [47.8‐87.5]) and showed all the maximum observed inhibition activity in the sVNT (98%), without overlaps between groups. There was a trend for higher responses in those with a more distant infection, although not statistically significant. The relative antibody increase after dose 2 was significantly higher among naïve individuals (25‐fold), but antibody levels remained below that of seropositives. Conclusions Compared with naïve individuals, seropositives after natural SARS‐CoV‐2 infection presented with a substantially higher antibody response already after dose 1 of BNT162b2, as measured by two CE‐marked in vitro diagnostic tests and a sVNT. These results should stimulate discussion and research on whether individuals after previous SARS‐CoV‐2 infection would benefit from a two‐part vaccination schedule or whether these currently much‐needed second doses could be saved.

BackgroundReliable quantification of the antibody response to SARS-CoV-2 vaccination is highly relevant for identifying possible vaccine failure and estimating the time of protection. Therefore, we aimed to evaluate the performance of five different Anti-SARS-CoV-2 antibody assays regarding the quantification of anti-spike (S) antibodies induced after a single dose of BNT162b2.MethodsSera of n=69 SARS-CoV-2 naïve individuals 21±1 days after vaccination with BNT162b2 (Pfizer/BioNTech) were tested using the following quantitative SARS-CoV-2 antibody assays: Roche S total antibody, DiaSorin trimeric spike IgG, DiaSorin S1/S2 IgG, Abbott II IgG, and Serion/Virion IgG. Test agreement was assessed by Passing-Bablok regression. Results were further compared to the percent inhibition calculated from a surrogate virus neutralization test (sVNT) by correlation and ROC (receiver-operating-characteristics) analysis.ResultsIndividual values were distributed over several orders of magnitude for all assays evaluated. Although the assays were in good overall agreement (ρ=0.80-0.94), Passing-Bablok regression revealed systematic and proportional differences, which could not be eliminated by converting the results to BAU/mL as suggested by the manufacturers. 7 (10%) individuals had a negative sVNT results (i.e. ConclusionsAlthough all assays evaluated showed good correlation, readings from different assays were not interchangeable, even when converted to BAU/mL using the WHO international standard for SARS-CoV-2 immunoglobulin. This highlights the need for further standardization of SARS-CoV-2 serology.

In an aging society, late‐life depression has become an increasing problem. There is evidence that physical activity ameliorates depressive symptoms and increases the quality of life (QoL). However, the underlying mechanisms are still poorly understood. Myokines are molecules secreted in response to muscle contraction. Some of them can cross the blood‐brain barrier, making them promising candidates for mediating the beneficial effects of physical activity on mood. The present study aims to compare circulating myokine levels to depression/QoL in older athletes and controls. 55 athletes, 57 controls >59 years were enrolled. The assessment included ergometry, magnetic resonance imaging, blood withdrawal, and neuropsychological testing. Serum interleukin‐6 (IL‐6), irisin, brain‐derived neurotrophic factor (BDNF), kynurenine, and cathepsin B were analyzed and compared to surrogates of depression and quality of life. Athletes presented with higher levels of Cathepsin B. Among controls, all myokines but irisin were associated with age. Also, among controls, kynurenine and IL‐6 correlated inversely with specific dimensions of quality of life questionnaires, and IL‐6 further with depressive symptoms and decreased physical performance. No such associations could be found among athletes. Irisin levels were inversely associated with mild depression and low‐grade white matter‐lesions in the brain and predicted impaired QoL. The circulating levels of several myokines/muscle activity‐related factors appear to be associated with depressive symptoms and impaired QoL among older adults. However, in athletes, some of these connections seem ameliorated, suggesting additional stressors (as f.e. age) or a different pathomechanism among athletes., Impact Statement Physical exercise positively affects mood and quality of life, however, the underlying mechanisms are not yet fully understood. Myokines are released in response to muscle contraction. They might be promising candidates for mediating muscle‐brain‐crosstalk. Our results suggest that basal kynurenine and interleukin‐6 levels are correlated with poor quality of life and/or depressive symptoms. In contrast, low irisin levels were associated with mild depression and low‐grade white matter‐hyperintensities in brain MRIs and they predicted impaired quality of life.

Neurofilament light chain (NfL) has emerged as a biomarker of neuroaxonal damage in several neurologic conditions. With increasing availability of fourth-generation immunoassays detecting NfL in blood, aspects of pre-analytical stability of this biomarker remain unanswered. This study investigated NfL concentrations in serum and plasma samples of 32 patients with neurological diagnoses using state of the art Simoa technology. We tested the effect of delayed freezing of up to 7 days and statistically determined stability and validity of measured concentrations. We found concentrations of NfL in serum and plasma to remain stable at room temperature when processing of samples is delayed up to 7 days (serum: mean absolute difference 0.9 pg/mL, intraindividual variation 1.2%; plasma: mean absolute difference 0.5 pg/mL, intraindividual variation 1.3%). Consistency of these results was nearly perfect for serum and excellent for plasma (intraclass correlation coefficients 0.99 and 0.94, respectively). In conclusion, the soluble serum and plasma NfL concentration remains stable when unprocessed blood samples are stored up to 7 days at room temperature. This information is essential for ensuring reliable study protocols, for example, when shipment of fresh samples is needed.

Background Serological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems is often poor, leaving room for false positive and false negative results. However, conventional methods used to increase specificity decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the "Sensitivity Improved Two-Test" or "SIT2" algorithm. Methods SIT2 involves confirmatory re-testing of samples with results falling in a predefined retesting-zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT2 to single tests and orthogonal testing (OTA) in an Austrian cohort (1,117 negative, 64 post-COVID positive samples) and validated the algorithm in an independent British cohort (976 negatives, 536 positives). Results The specificity of SIT2 was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT2 when compared to single tests or OTA. SIT2 allowed correct identification of infected individuals even when a live virus neutralization assay could not detect antibodies. Compared to single testing or OTA, SIT2 significantly reduced total test errors to 0.46% (0.24-0.65) or 1.60% (0.94-2.38) at both 5% or 20% seroprevalence. Conclusion For SARS-CoV-2 serology, SIT2 proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases.

Background In the context of the COVID-19 pandemic, numerous new serological test systems for the detection of anti-SARS-CoV-2 antibodies rapidly have become available. However, the clinical performance of many of these is still insufficiently described. Therefore, we compared 3 commercial CE-marked, SARS-CoV-2 antibody assays side by side. Methods We included a total of 1154 specimens from pre-COVID-19 times and 65 samples from COVID-19 patients (≥14 days after symptom onset) to evaluate the test performance of SARS-CoV-2 serological assays by Abbott, Roche, and DiaSorin. Results All 3 assays presented with high specificities: 99.2% (98.6–99.7) for Abbott, 99.7% (99.2–100.0) for Roche, and 98.3% (97.3–98.9) for DiaSorin. In contrast to the manufacturers’ specifications, sensitivities only ranged from 83.1% to 89.2%. Although the 3 methods were in good agreement (Cohen’s Kappa 0.71–0.87), McNemar tests revealed significant differences between results obtained from Roche and DiaSorin. However, at low seroprevalences, the minor differences in specificity resulted in profound discrepancies of positive predictive values at 1% seroprevalence: 52.3% (36.2–67.9), 77.6% (52.8–91.5), and 32.6% (23.6–43.1) for Abbott, Roche, and DiaSorin, respectively. Conclusion We found diagnostically relevant differences in specificities for the anti-SARS-CoV-2 antibody assays by Abbott, Roche, and DiaSorin that have a significant impact on the positive predictive values of these tests.

Small extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers for systemic disease; however interpretation is complicated by release of sEV ex vivo after blood taking. To quantify the problem and devise ways to minimise it, we characterised sEV in paired serum, plasma and platelet poor plasma (PPP) samples from healthy donors. Immunoblotting showed twofold greater abundance of CD9 in sEV fractions from fresh serum than from fresh plasma or PPP. MACSPlex confirmed this, and showed that proteins expressed on platelet sEV, either exclusively (CD41b, CD42a and CD62P) or more widely (HLA-ABC, CD24, CD29 and CD31) were also twofold more abundant; by contrast non-platelet proteins (including CD81) were no different. Storage of plasma (but not serum) increased abundance of platelet and selected leukocyte sEV proteins to at least that of serum, and this could be recapitulated by activating cells in fresh plasma by Ca2+, an effect abrogated in PPP. This suggests that a substantial proportion of sEV in serum and stored plasma were generated ex vivo, which is not the case for fresh plasma or PPP. Thus we provide strategies to minimise ex vivo sEV generation and criteria for identifying those that were present in vivo.