Patient and blood samples

Patients were recruited from the John Radcliffe Hospital in Oxford, UK, between March and May 2020 by identification of patients hospitalised during the SARS-CoV-2 pandemic and recruited into the Sepsis Immunomics project [Oxford REC C, reference:19/SC/0296] ISARIC/WHO Clinical Characterization Protocol for Severe Emerging Infections [Oxford REC C, reference 13/SC/0149]. Time between onset of symptoms and sampling were known for all patients and if labeled as convalescent patients were sampled at least 28 days from the start of their symptoms. Written informed consent was obtained from all patients. All patients were confirmed to have tested positive for SARS-CoV-2 using the reverse transcriptase polymerase chain reaction (RT-PCR) from an upper respiratory tract (nose/throat) swab tested in accredited laboratories. The degree of severity was identified as a mild, severe or critical infection according to recommendations from the World Health Organization. Severe infection was defined for COVID-19 confirmed patients with one of the following conditions: respiratory distress with RR > 30/min; blood oxygen saturation < 93%; arterial oxygen partial pressure (PaO2) / fraction of inspired O2 (FiO2) < 300 mmHg; and critical infection was defined as respiratory failure requiring mechanical ventilation or shock; or other organ failures requiring admission to ICU. Comparator samples from healthcare workers or epidemiologically detected early clusters with confirmed SARS-CoV-2 infection who all had mild non-hospitalised disease were collected under the Gastro-intestinal illness in Oxford: COVID sub study [Sheffield REC, reference: 16/YH/0247].

Blood samples were collected and separated into plasma by centrifugation at 500 g for 10 mins. Plasma was removed from the uppermost layer and stored at −80°C. The PBMC layer was then gently suspended in the remaining plasma and RPMI media, and then isolated by Ficoll-Hypaque gradient centrifugation. All PBMC samples were stored in liquid nitrogen until use.