A validated protocol to UV-inactivate SARS-CoV-2 and herpesvirus-infected cells.

Downstream analysis of virus-infected cell samples, such as reverse transcription polymerase chain reaction (RT PCR) or mass spectrometry, often needs to be performed at lower biosafety levels than their actual cultivation, and thus the samples require inactivation before they can be transferred. Common inactivation methods involve chemical crosslinking with formaldehyde or denaturing samples with strong detergents, such as sodium dodecyl sulfate. However, these protocols destroy the protein quaternary structure and prevent the analysis of protein complexes, albeit through different chemical mechanisms. This often leads to studies being performed in over-expression or surrogate model systems. To address this problem, we generated a protocol that achieves the inactivation of infected cells through ultraviolet (UV) irradiation. UV irradiation damages viral genomes and crosslinks nucleic acids to proteins but leaves the overall structure of protein complexes mostly intact. Protein analysis can then be performed from intact cells without biosafety containment. While UV treatment protocols have been established to inactivate viral solutions, a protocol was missing to inactivate crude infected cell lysates, which heavily absorb light. In this work, we develop and validate a UV inactivation protocol for SARS-CoV-2, HSV-1, and HCMV-infected cells. A fluence of 10,000 mJ/cm2 with intermittent mixing was sufficient to completely inactivate infected cells, as demonstrated by the absence of viral replication even after three sequential passages of cells inoculated with the treated material. The herein described protocol should serve as a reference for inactivating cells infected with these or similar viruses and allow for the analysis of protein quaternary structure from bona fide infected cells.

Anti-SARS-CoV-2 antibodies in breast milk during lactation after infection or vaccination: A cohort study.

Breast milk is a pivotal source to provide passive immunity in newborns over the first few months of life. Very little is known about the antibody transfer levels over the period of breastfeeding. We conducted a prospective study in which we evaluated concentrations of anti-SARS-CoV-2 Spike IgA and RBD IgG/M/A antibodies in maternal serum and breast milk over a duration of up to 6 months after delivery. We compared antibody levels in women with confirmed COVID-19 infection during pregnancy (n = 16) to women with prenatal SARS-CoV-2 vaccination (n = 5). Among the recovered women, n = 7 (44%) had been vaccinated during the lactation period as well. We observed intraindividual moderate positive correlations between antibody levels in maternal serum and breast milk (r = 0.73, p-value<0.0001), whereupon the median levels were generally higher in serum. Anti-RBD IgA/M/G transfer into breast milk was significantly higher in women recovered from COVID-19 and vaccinated during lactation (35.15 AU/ml; IQR 21.96-66.89 AU/ml) compared to the nonvaccinated recovered group (1.26 AU/ml; IQR 0.49-3.81 AU/ml), as well as in the vaccinated only group (4.52 AU/ml; IQR 3.19-6.23 AU/ml). Notably, the antibody level in breast milk post SARS-CoV-2 infection sharply increased following a single dose of vaccine. Breast milk antibodies in all groups showed neutralization capacities against an early pandemic SARS-CoV-2 isolate (HH-1) and moreover, also against the Omicron variant, although with lower antibody titer. Our findings highlight the importance of booster vaccinations especially after SARS-CoV-2 infection in pregnancy in order to optimize protection in mother and newborn.

Fcγ-Receptor-Based Enzyme-Linked Immunosorbent Assays for Sensitive, Specific, and Persistent Detection of Anti-SARS-CoV-2 Nucleocapsid Protein IgG Antibodies in Human Sera.

Sensitive and specific serological tests are mandatory for epidemiological studies evaluating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence as well as coronavirus disease 2019 (COVID-19) morbidity and mortality rates. The accuracy of results is challenged by antibody waning after convalescence and by cross-reactivity induced by previous infections with other pathogens. By employing a patented platform technology based on capturing antigen-antibody complexes with a solid-phase-bound Fcγ receptor (FcγR) and truncated nucleocapsid protein as the antigen, two SARS-CoV-2 IgG enzyme-linked immunosorbent assays (ELISAs), featuring different serum and antigen dilutions, were developed. Validation was performed using a serum panel comprising 213 longitudinal samples from 35 COVID-19 patients and a negative-control panel consisting of 790 pre-COVID-19 samples from different regions of the world. While both assays show similar diagnostic sensitivities in the early convalescent phase, ELISA 2 (featuring a higher serum concentration) enables SARS-CoV-2 IgG antibody detection for a significantly longer time postinfection (≥15 months). Correspondingly, analytical sensitivity referenced to indirect immunofluorescence testing (IIFT) is significantly higher for ELISA 2 in samples with a titer of ≤1:640; for high-titer samples, a prozone effect is observed for ELISA 2. The specificities of both ELISAs were excellent not only for pre-COVID-19 serum samples from Europe, Asia, and South America but also for several challenging African sample panels. The SARS-CoV-2 IgG FcγR ELISAs, methodically combining antigen-antibody binding in solution and isotype-specific detection of immune complexes, are valuable tools for seroprevalence studies requiring the (long-term) detection of anti-SARS-CoV-2 IgG antibodies in populations with a challenging immunological background and/or in which spike-protein-based vaccine programs have been rolled out.

Clinical efficacy and in vitro neutralization capacity of monoclonal antibodies for severe acute respiratory syndrome coronavirus 2 delta and omicron variants.

We aimed to provide in vitro data on the neutralization capacity of different monoclonal antibody (mAb) preparations against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delta and omicron variant, respectively, and describe the in vivo RNA kinetics of coronavirus disease 2019 (COVID-19) patients treated with the respective mAbs. Virus neutralization assays were performed to assess the neutralizing effect of the mAb formulations casirivimab/imdevimab and sotrovimab on the SARS-CoV-2 delta and omicron variant. Additionally, respiratory tract SARS-CoV-2 RNA kinetics are provided for 25 COVID-19 patients infected with either delta variant (n = 18) or omicron variant (n = 7) treated with the respective mAb formulations during their hospital stay. In the virus neutralization assay, sotrovimab exhibits neutralizing capacity at therapeutically achievable concentrations against the SARS-CoV-2 delta and omicron variant. In contrast, casivirimab/imdevimab had neutralizing capacity against the delta variant but failed neutralization against the omicron variant except for a very high concentration above the currently recommended therapeutic dosage. In patients with delta variant infections treated with casivirimab/imdevimab, we observed a rapid decrease of respiratory viral RNA at day 3 after mAb therapy. In contrast, no such prompt decline was observed in patients with delta variant or omicron variant infections receiving sotrovimab.

Longitudinal detection of SARS-CoV-2-specific antibody responses with different serological methods.

Serological testing for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is used to detect ongoing or past SARS-CoV-2 infections. To study the kinetics of anti-SARS-CoV-2 antibodies and to assess the diagnostic performances of eight serological assays, we used 129 serum samples collected on known days post symptom onset (dpso) from 42 patients with polymerase chain reaction-confirmed coronavirus disease 2019 (COVID-19) and 54 serum samples from healthy blood donors, and children infected with seasonal coronaviruses. The sera were analyzed for the presence of immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) antibodies using indirect immunofluorescence testing (IIFT) based on SARS-CoV-2-infected cells. They were further tested for antibodies against the S1 domain of the SARS-CoV-2 spike protein (IgG, IgA) and against the viral nucleocapsid protein (IgG, IgM) using enzyme-linked immunosorbent assays. The assay specificities were 94.4%-100%. The sensitivities varied largely between assays, reflecting their respective purposes. The sensitivities of IgA and IgM assays were the highest between 11 and 20 dpso, whereas the sensitivities of IgG assays peaked between 20 and 60 dpso. IIFT showed the highest sensitivities due to the use of the whole SARS-CoV-2 as substrate and provided information on whether or not the individual has been infected with SARS-CoV-2. Enzyme-linked immunosorbent assays provided further information about both the prevalence and concentration of specific antibodies against selected antigens of SARS-CoV-2.

Cover Image, Volume 93, Number 10, October 2021

Front Cover Caption: The cover image is based on the Research Article Longitudinal detection of SARS-CoV-2-specific antibody responses with different serological methods by Petra Emmerich et al., https://doi.org/10.1002/jmv.27113.

Limited specificity of commercially available SARS-CoV-2 IgG ELISAs in serum samples of African origin.

OBJECTIVES: Specific serological tests are mandatory for reliable SARS-CoV-2 diagnostics and seroprevalence studies. Here, we assess the specificities of four commercially available SARS-CoV-2 IgG ELISAs in serum/plasma panels originating from Africa, South America, and Europe. METHODS: 882 serum/plasma samples collected from symptom-free donors before the COVID-19 pandemic in three African countries (Ghana, Madagascar, Nigeria), Colombia, and Germany were analysed with three nucleocapsid-based ELISAs (Euroimmun Anti-SARS-CoV-2-NCP IgG, EDI™ Novel Coronavirus COVID-19 IgG, Mikrogen recomWell SARS-CoV-2 IgG), one spike/S1-based ELISA (Euroimmun Anti-SARS-CoV-2 IgG), and in-house common cold CoV ELISAs. RESULTS: High specificity was confirmed for all SARS-CoV-2 IgG ELISAs for Madagascan (93.4-99.4%), Colombian (97.8-100.0%), and German (95.9-100.0%) samples. In contrast, specificity was much lower for the Ghanaian and Nigerian serum panels (Ghana: NCP-based assays 77.7-89.7%, spike/S1-based assay 94.3%; Nigeria: NCP-based assays 39.3-82.7%, spike/S1-based assay 90.7%). 15 of 600 African sera were concordantly classified as positive in both the NCP-based and the spike/S1-based Euroimmun ELISA, but did not inhibit spike/ACE2 binding in a surrogate virus neutralisation test. IgG antibodies elicited by previous infections with common cold CoVs were found in all sample panels, including those from Madagascar, Colombia, and Germany and thus do not inevitably hamper assay specificity. Nevertheless, high levels of IgG antibodies interacting with OC43 NCP were found in all 15 SARS-CoV-2 NCP/spike/S1 ELISA positive sera. CONCLUSIONS: Depending on the chosen antigen and assay protocol, SARS-CoV-2 IgG ELISA specificity may be significantly reduced in certain populations probably due to interference of immune responses to endemic pathogens like other viruses or parasites.

Mütter-Screening in einem COVID-19-Niedrig-Pandemiegebiet: Bestimmung SARS-CoV-2-spezifischer Antikörper bei 401 Rostocker Müttern mittels ELISA und Immunfluoreszenz-Bestätigungstest

BACKGROUND: In children, the infection with SARS-CoV-2, the cause of COVID-19, tends to be clinically inapparent more often or less severe than in adults. The spread of this infection from children poses a danger to vulnerable adults. Therefore, child care institutions and schools currently are widely closed. METHODS: Since the status of infection tends to be congruent in mothers and their children, we tested 401 mothers of children between 1 and 10 years in the city of Rostock (State of Mecklenburg-Westpomerania, northeast of Germany), for the presence of RNA of SARS-CoV-2 in throat swabs, and of antibodies against SARS-CoV-2 in serum. RESULTS: In none of the mothers tested, RNA of this virus was detected in the throat swab. In the ELISA test, IgG antibodies were positive in one serum sample, IgA antibodies were positive in 11, and borderline in 3 serum samples. All 401 sera were negative in the indirect immunofluorescence test (IIFT) with FITC-labeled IgG, IgA, und IgM antibodies. CONCLUSION: At the time of this study, neither SARS-CoV-2 RNA, nor specific antibodies against SARS-CoV-2 were detectable in the mothers tested in Rostock.

[Screening of Mothers in a COVID-19 Low-Prevalence Region: Determination of SARS-CoV-2 Antibodies in 401 Mothers from Rostock by ELISA and Confirmation by Immunofluorescence]. / Mütter-Screening in einem COVID-19-Niedrig-Pandemiegebiet: Bestimmung SARS-CoV-2-spezifischer Antikörper bei 401 Rostocker Müttern mittels ELISA und Immunfluoreszenz-Bestätigungstest.

BACKGROUND: In children, the infection with SARS-CoV-2, the cause of COVID-19, tends to be clinically inapparent more often or less severe than in adults. The spread of this infection from children poses a danger to vulnerable adults. Therefore, child care institutions and schools currently are widely closed. METHODS: Since the status of infection tends to be congruent in mothers and their children, we tested 401 mothers of children between 1 and 10 years in the city of Rostock (State of Mecklenburg-Westpomerania, northeast of Germany), for the presence of RNA of SARS-CoV-2 in throat swabs, and of antibodies against SARS-CoV-2 in serum. RESULTS: In none of the mothers tested, RNA of this virus was detected in the throat swab. In the ELISA test, IgG antibodies were positive in one serum sample, IgA antibodies were positive in 11, and borderline in 3 serum samples. All 401 sera were negative in the indirect immunofluorescence test (IIFT) with FITC-labeled IgG, IgA, und IgM antibodies. CONCLUSION: At the time of this study, neither SARS-CoV-2 RNA, nor specific antibodies against SARS-CoV-2 were detectable in the mothers tested in Rostock.