Chapter 8 244 SARS-CoV-2 binding IgG antibody levels Levels of Immunoglobulin G (IgG) binding to SARS-CoV-2 receptor-binding domain (RBD) and spike (S) proteins of wild type virus (Wu-1) were determined using a custom luminex assay as described previously[11, 12]. In summary, proteins were produced in HEK293F cells (Invitrogen) and purified from the cell culture supernatant using affinity chromatography with NiNTA agarose beads (Qiagen). Proteins were covalently coupled to luminex magplex beads using a two-step carbodiimide reaction. Beads were then incubated overnight with 1:100,000 diluted serum followed by detection with goatanti-human IgG-PE (Southern Biotech) on a Magpix (Luminex) as the mean fluorescent intensity (MFI). Neutralisation assays Pseudovirus neutralization assay was performed as previously described[13]. Briefly, HEK293T/ACE2 cells[14] were seeded in poly-L-lysine pre-coated 96-well plates. The next day, heat-inactivated 1:100 diluted sera were 3-fold serially diluted and mixed in a 1:1 ratio with pseudovirus Wu-1 D614G (WT)[14]. After 1-hour incubation at 37°C the mixtures were added to the cells and incubated for 48 hours at 37°C. The luciferase activity in cell lysates was measured using the Nano-Glo Luciferase Assay System (Promega) and GloMax system (Turner BioSystems). The 50% inhibitory dilution (ID50) titers were determined as the serum dilution at which infectivity was inhibited by 50% using a non-linear regression curve fit (GraphPad Prism software version 8.3). Statistical analysis Baseline socio-demographic and clinical characteristics of participants with at least 3 months of follow-up were compared between those who developed PASC and those who recovered from all COVID-19 symptoms within 3 months of illness onset. We first evaluated the effect of COVID-19 vaccination on the mean numbers of PASC symptoms reported. Among participants with PASC, we matched vaccinated and unvaccinated follow-up intervals according to participants’ age group (<45 years; 4565 years; 65+ years), sex (male/female), BMI (obese or not) and time since illness onset (in months). This was achieved by 1:1 exact matching the month in which a participant received their first vaccination to a participant who remained unvaccinated for at least one month following the matched time-point, using a coarsened exact matching (CEM) approach[15]. Follow-up was then censored at lost to follow-up, last cohort visit or at
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