Ethics statement
All human serum samples were obtained with written informed consent from the participants (2020/ETH00964; 2018/ETH00145; 2021/ETH00180). Primary bronchial epithelial cells (pBEC) were provided by P. A. B. Wark (University of Newcastle), and originally obtained from one healthy non-smoking donor (73-year-old female) during bronchoscopy, with written informed consent. Experiments were conducted with approval from the University of Newcastle Safety Committee (Safety REF# 25/2016 and R5/2017). All participants underwent fibre-optic bronchoscopy in accordance with standard guideline47. All primary isolates used in this study were obtained from de-identified remnant diagnostic swabs that had completed all diagnostic testing under approval by the New South Wales Chief Health Officer following independent scientific review (2021/NSWCHO H21/126831) and as outlined in the ADAPT ethics protocol (2020/ETH00964).
Cell culture
HEK293T cells (Thermo Fisher, R70007), HEK293T derivatives and VeroE6-TMPRSS2 (CellBank Australia, JCRB1819) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11995073) with 10% fetal bovine serum (FBS) (Sigma, F423-500). VeroE6 cells (ATCC CRL-1586) were cultured in Minimum Essential Medium (MEM; Sigma, M4655) with 10% FBS and 1% penicillin-streptomycin (Gibco, 15140122). pBEC cultures were grown and differentiated until confluent in complete Bronchial Epithelial Cell Growth Basal Medium (Lonza, CC-3171) before use for air–liquid interface experiments. All cells were cultured and incubated at 37 °C, 5% CO2 and >90% relative humidity, unless otherwise indicated.
Participants and patient samples
Three cohorts of human study participants were considered for studying neutralization of viral variants. The ADAPT cohort is composed of RT–qPCR-confirmed convalescent individuals recruited during 2020 in Australia23, many of which have now been vaccinated. Of the >200 ADAPT participants, representative donors were chosen on the basis of serum neutralization titres against the Clade B virus in this study. A second cohort was composed of 24 healthy adult vaccine recipients who received the BNT162b2 vaccine in 2021. A third cohort was rapidly formed at the time of Omicron arrival in Australia and consisted of 25 healthcare workers who were 4 weeks post their third BNT162b2 vaccine dose. Median age, interquartile age range and female to male ratios are outlined for each group in Supplementary Tables 1–9.
Monoclonal immunoglobulin products
Monoclonal antibodies were provided by the Garvan Institute of Medical Research, Australia. Briefly, DNA sequences encoding the variable domain sequences of the therapeutic monoclonal antibodies Sotrovimab, Casirivimab, Imdevimab, Bamlanivimab, Cilgavimab and Tixagevimab were generated by gene synthesis, cloned into human IgG1 expression vectors and produced in ExpiCHO cells (Thermo Fisher, A29133)48. After production in ExpiCHO cells, monoclonal antibodies were characterized for binding to recombinant RBD by biolayer interferometry and for neutralization of live early clade (A2.2) SARS-CoV-2 virus48.
Polyclonal immunoglobulin preparations and anti-SARS-CoV-2 hyperimmune globulin
A CoVIg-19 Plasma Alliance was formed in 2020 between major plasma pharmaceuticals including CSL, Takeda, Octapharma and Sanquin, with an aim to develop a COVID-19 immunoglobulin therapy. As part of that initiative, CSL Behring manufactured anti-SARS-CoV-2 hyperimmune globulin (CoVIg). Approximately 5,000 convalescent donor plasma units were collected between September and October 2020, exclusively from SARS-CoV-2 convalescent donors. After COVID-19 confirmation49, the immunoglobulin was purified using the licensed and fully validated immunoglobulin manufacturing process used for Privigen50, notionally similar to others51. Five intravenous immunoglobulin (IVIG) lots (Poly IgG 1033, 4850, 7450, 0301, 0723) manufactured using the Privigen process described by Stucki et al.50 included US plasma collected by plasmapheresis from a mixture of donors vaccinated with SARS-CoV-2 mRNA vaccines, convalescent and non-convalescent donors (source plasma, n between 9,495–23,667 per batch). The majority of donations were collected between April and June 2021 (Supplementary Fig. 5). The Plasma Alliance control was obtained from CSL Behring and the WHO international reference standard for SARS-CoV-2 neutralization (NIBSC 20/136) was obtained from NIBSC25.
Generation of HEK293T-ACE2-TMPRSS2 cells (clone HAT-24)
HEK293T cells stably expressing human ACE2 and TMPRSS2 were generated by lentiviral transductions as previously described23. Briefly, the open reading frames for hACE2 (Addgene, 1786) and hTMPRSS2a (IDT, synthetic gene fragment) were cloned into lentiviral expression vectors pRRLsinPPT.CMV.GFP.WPRE52 and pLVX-IRES-ZsGreen (Clontech, 632187), respectively. For ACE2 cloning, Age1/Bsrg1 cut sites were used to replace GFP with ACE2, while hTMPRSS2a was cloned into pLVX-IRES-ZsGreen using EcoR1/XhoI restriction sites. Lentiviral particles expressing the above genes were produced by co-transfecting expression plasmids individually with a 2nd generation lentiviral packaging construct psPAX2 (courtesy of Dr Didier Trono through NIH AIDS repository) and VSVG plasmid pMD2.G (Addgene, 2259) in HEK293T producer cells using polyethyleneimine as previously described53. Virus supernatant was collected 72 h post-transfection, pre-cleared of cellular debris and centrifuged at 28,000 × g for 90 min at 4 °C to generate concentrated virus stocks. Two successive rounds of lentiviral transductions were then performed on HEK293T cells to generate ACE2-TMPRSS2 HEK293T cells. Clonal selection led to the identification of a highly permissive clone (HAT-24), which was then used in subsequent experiments23.
Viral isolation from primary specimens
Respiratory specimens were collected and stored at 4 °C for same-day diagnostic RT–qPCR (Allplex SARS-CoV-2 Assay, Seegene). Specimens positive for SARS-CoV-2 were then frozen at −80 °C within 24 h of collection, and later transported to a certified BSL-3 facility for primary isolate propagation. Thawed viral eluate was sterile-filtered through 0.22 µm column filters (Merck, UFC30GVOS) at 10,000 × g for 5 min and then serially diluted (3-fold series) in quadruplicate. Viral dilutions were added to HAT-24 cells seeded in 96-well plates at 104 cells per well (final volume, 100 µl). Plates were incubated at 37 °C and monitored by brightfield microscopy every 24 h using high-content microscopy. Once extensive CPE became evident in at least 2 dilutions (Fig. 1e); the cells and supernatant from these cultures were collected and cleared from debris by centrifugation at 2,000 × g for 5 min, aliquoted and stored at −80 °C (passage 1). For further expansion, 300 μl of passage 1 virus including infected cells was used to resuspend a pellet of 0.5 × 106 VeroE6 cells in suspension. After incubation for 30 min at 37 °C, the co-culture was transferred to a 6-well plate with 2 ml of MEM-2% FBS medium per well. The resulting supernatant was collected after 48 to 72 h (when visible and extensive CPE was observed), cleared by centrifugation as above and stored at –80 °C (passage 2). A final larger expansion step (passage 3) was conducted by resuspending 2 × 107 VeroE6 cells in 500 μl of diluted passage 2 virus (MOI = 0.05), incubating at 37 °C for 48 h, and clearing and storing the supernatant at −80 °C as above. Shorter expansions with higher titres were also achieved in pre-Omicron variants by infecting the VeroE6-TMPRSS2 cell line with the same MOI but collecting cells at 24 h post infection. Sequence identity and integrity were confirmed for both passages 1 and 3 virus via whole-genome viral sequencing as described further below.
Titration of primary nasopharyngeal swabs
Viral eluates from primary specimens were thawed and sterile-filtered as indicated above, and coded before leaving the diagnostic laboratory. Samples were then diluted in 96-well plates (3-fold series, in quadruplicate) and 40 µl of each dilution were transferred to an equal volume of freshly plated HAT-24 cells seeded in 384-well plates (CLS3985, Corning) at 1.6 × 104 cells per well in DMEM-5% FBS medium. After incubation at 37 °C for 72–96 h, whole wells were imaged by high-content brightfield microscopy, and images were binarily scored by two independent experienced operators for CPE (+ or −) to determine the viral end-point titre (that is, last dilution containing at least one ‘+’ well).
Whole-genome viral sequencing
Clinical respiratory specimens positive by diagnostic SARS-CoV-2 PCR were sequenced using a combination or Nanopore single-molecule sequencing and amplicon-based Illumina sequencing approach, as previously described11. Consensus SARS-CoV-2 genomes have been uploaded to GISAID (www.gisaid.org) and are publicly available as indicated in Supplementary Table 1.
Titration of expanded viral stocks
For overnight titrations (20 h format), HAT-24 cells were trypsinized, resuspended in DMEM-5% FBS medium with Hoechst-33342 live nuclear dye (Invitrogen, R37605) at 5% v/v and seeded in 384-well plates (Corning, CLS3985) at 1.6 × 104 cells per well. For traditional titrations (72 h format), HAT-24 or VeroE6 cells were seeded at 5 × 103 cells per well in DMEM-5% FBS or MEM-2% FBS, respectively, and stained with Hoechst-33342 only after the 72 h virus incubation. SARS-CoV-2 viral stocks were serially diluted (5-fold series) in cell culture medium in octuplicate and then 40 μl of viral dilution were added to an equal volume of the freshly plated cells. Plates were incubated for either 20 h (rapid overnight titration) or 72 h (traditional method) before the entire plate area was imaged on an InCell Analyzer HS2500 high-content microscope (Cytiva). Brightfield images were visually inspected…