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.

Vero (ATCC CCL-81) cells and Vero-furin cells (Mukherjee et al., 2016) were cultured at 37°C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, and 100 U/ml of penicillin–streptomycin Spike ectodomain, human mAbs and Fabs were expressed in HEK293T cells cultured in FreeStyle 293 Expression Medium (12338018, ThermoFisher) at 37°C with 8% CO2. Nucleoprotein was expressed using 2-L cultures of Rosettagami2(DE3)pLysS bacteria (Novagen) in terrific broth medium containing 40 mg/L kanamycin, at 15°C for 40 hr following induction with Isopropyl β-D-1-thiogalactopyranoside (1mM final concentration, Meridian Bioscience). For ACE2 and RBD, transient expression used Expi293F cells (Thermo Fisher, Cat# A14527) grown in Expi293 Expression Medium (Thermo Fisher Cat# A1435103) in suspension with 8% CO2 at 30 or 37°C and shaking at 130 rpm. For production of Spike protein for structural analysis, HEKExpi293F cells (Thermo Fisher Scientific) were transfected with the construct together with a phiC31 integrase expression plasmid and grown in adhesion roller bottles with the high glucose DMEM (Sigma) with 2% FBS for 6 days at 30°C. His-tagged RBD for structural analysis was expressed in a stable HEK293S cell line cultured in DMEM (high glucose, Sigma) supplemented with 10% FBS (Invitrogen), 1 mM glutamine and 1x non-essential amino acids at 37 °C. Cells were transferred to roller bottles (Greiner) and cultured in DMEM supplemented with 2% FBS, 1 mM glutamine and 1x non-essential amino acids at 30 °C for 10 days for protein expression. For plaque assays Vero-furin cells (Mukherjee et al.,2016) were cultured at 37°C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, and 100 U/ml of penicillin–streptomycin.

SARS-CoV-2/human/AUS/VIC01/2020 (Caly et al., 2020) was grown in Vero (ATCC CCL-81) cells. Virus containing supernatant was spun at 2000 rpm at 4°C before being stored at −80°C. Viral titers were determined by a focus-forming assay on Vero cells. For mouse experiments, the 2019n-CoV/USA_WA1/2020 isolate of SARS-CoV-2 was obtained from the US Centers for Disease Control (CDC). Infectious stocks were propagated by inoculating Vero CCL81 cells and collecting supernatant upon observation of cytopathic effect; debris was removed by centrifugation and passage through a 0.22 μm filter. Supernatant was aliquoted and stored at −80°C.

Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381–01). Virus inoculations were performed under anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering.

Heterozygous K18-hACE C57BL/6J mice (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained from The Jackson Laboratory. Seven to eight-week-old male and female animals were inoculated with 103 PFU of SARS-CoV-2 via intranasal administration.

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