ABSTRACT
BackgroundLong-term persistence of antibodies against SARS-CoV-2, particularly the SARS-CoV-2 Spike Trimer, determines individual protection against infection and potentially viral spread. The quality of childrens natural humoral immune response following SARS-CoV-2 infection is yet incompletely understood but crucial to guide pediatric SARS-CoV-2 vaccination programs. MethodsIn this prospective observational multi-center cohort study, we followed 328 households, consisting of 548 children and 717 adults, with at least one member with a previous laboratory-confirmed SARS-CoV-2 infection. The serological response was assessed at 3-4 months and 11-12 months after infection using a bead-based multiplex immunoassay for 23 human coronavirus antigens including SARS-CoV-2 and its Variants of Concern (VOC) and endemic human coronaviruses (HCoVs), and additionally by three commercial SARS-CoV-2 antibody assays. ResultsOverall, 33.76% of SARS-CoV-2 exposed children and 57.88% adults were seropositive. Children were five times more likely to have seroconverted without symptoms compared to adults. Despite the frequently asymptomatic course of infection, children had higher specific antibody levels, and their antibodies persisted longer than in adults (96.22% versus 82.89% still seropositive 11-12 months post infection). Of note, symptomatic and asymptomatic infections induced similar humoral responses in all age groups. In symptomatic children, only dysgeusia was found as diagnostic indicator of COVID-19. SARS-CoV-2 infections occurred independent of HCoV serostatus. Antibody binding responses to VOCs were similar in children and adults, with reduced binding for the Beta variant in both groups. ConclusionsThe long-term humoral immune response to SARS-CoV-2 infection in children is robust and may provide long-term protection even after asymptomatic infection. (Study ID at German Clinical Trials Register: 00021521)
ABSTRACT
<p><b>OBJECTIVE</b>To study the sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells.</p><p><b>METHODS</b>Sensitivity and specificity of several common methods for apoptosis determination were evaluated (Apo2.7-expression, Annexin V-binding, TUNEL-reaction, poly-(ADP-ribose)-polymerase-(PARP) cleavage and single-stranded-DNA (ssDNA) staining). Apoptosis was induced by oxidative stress generated by hydrogen peroxide in 3 cultured cells types growing as adherent monolayer (MiaPaCa-2, Hep-G2 and human skin fibroblasts), necrosis was induced by depletion of cellular ATP using sodium azide. Cells positively stained by the respective apoptosis assay were quantified and alterations of cell morphology were monitored by fluorescence microscopy. The date was analyzed by one-way analysis of variance and significance test of correlation coefficient.</p><p><b>RESULTS</b>One hour after apoptosis induction significant cell fractions were positively stained for ssDNA (33% with MiaPaCa-2 cells, 35% with Hep-G2 cells, 56% with human skin fibroblasts). PARP-cleavage was less sensitive compared to the ssDNA-staining. Apo2.7-expression, Annexin V-binding and TUNEL-reaction were not applicable to detect early apoptosis induced by oxidative stress (below 2 hours), but were efficiently monitoring late apoptosis. Specificity of ssDNA-staining was complete with each cell type even 4 hs after induction of necrosis by the highest sodium azide concentration. In contrast, the same experimental conditions resulted in 50% - 90% positively stained necrotic cells by using Apo2.7-expression, TUNEL-reaction or Annexin V-binding. Surprisingly, specificity of PARP-cleavage was highly depending on the respective cell type.</p><p><b>CONCLUSIONS</b>Our study prove that among the five methods investigated only ssDNA-staining allowed to completely differentiate apoptosis from necrosis, and is thus suitable to reliably detect early as well as late apoptosis. Therefore, the ssDNA-staining may be used as reference method to clearly identify apoptosis induced by oxidative stress in adherent cells. The TUNEL-reaction, annexin-V-binding and Apo-2.7-expression may be used to quantify the number of apoptotic and necrotic cells especially at later stages but without discrimination of apoptosis and primary or secondary necrosis.</p>
