Platform for isolation and characterization of SARS-CoV-2 variants enables rapid characterization of Omicron in Australia

Genetically distinct variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged since the start of the COVID-19 pandemic. Over this period, we developed a rapid platform (R-20) for viral isolation and characterization using primary remnant diagnostic swabs. This, combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterization of all major SARS-CoV-2 variants circulating in Australia in 2021. Our platform facilitated viral variant isolation, rapid resolution of variant fitness using nasopharyngeal swabs and ranking of evasion of neutralizing antibodies. In late 2021, variant of concern Omicron (B1.1.529) emerged. Using our platform, we detected and characterized SARS-CoV-2 VOC Omicron. We show that Omicron effectively evades neutralization antibodies and has a different entry route that is TMPRSS2-independent. Our low-cost platform is available to all and can detect all variants of SARS-CoV-2 studied so far, with the main limitation being that our platform still requires appropriate biocontainment.

A s of the end of 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID- 19), has accounted for close to half a billion infections and millions of deaths worldwide (https://covid19. who.int/). Throughout the pandemic, continuous viral spread has led to the accumulation of many polymorphisms primarily within its Spike glycoprotein. Many changes drove higher levels of transmission [1][2][3][4] and successive waves of infection where several variants of concern (VOC) dominated. In 2021, Delta VOC globally dominated, supplanting all VOCs Alpha, Beta and Gamma (https://nextstrain.org/ncov/open/global).
In Australia, the combination of lockdowns and international border closures led to a very low level of community cases during 2020 5 . In many states, the only appearance of the virus was primarily through quarantine networks for returning overseas travellers [6][7][8] . New South Wales (NSW) over this time welcomed the majority of returning travellers entering Australia and during the period of December 2020 through to June 2021, all VOCs and the majority of variants under investigation (VUI) were detected through rapid whole-genome sequencing (WGS) [9][10][11] . Access to this network through research-diagnostics partnerships enabled the study of emerging variants from geographically diverse sites at a time where community spread in Australia was either absent or minimal. In this setting, this partnership provided a sentinel programme, where genomic detection, isolation and characterization enabled observations which then informed on the risk of key emerging variants.
Previous work generating various cell lines and the powerful use of reverse genetics has helped many aspects of the COVID-19 response [12][13][14] . Alongside these approaches, our aim was to enable our sentinel programme by developing a rapid, accessible and scalable set of platforms that led to sensitive viral isolation and subsequent characterization. The utility of our platforms was then tested stringently at several levels. First, we tested our platform in rapid isolation of many globally relevant variants isolated from swabs with low viral loads (Quantitative Polymerase Chain Reaction Cycle Threshold (qPCR Ct) >30). Second, we characterized variants against a range of humoral responses and therapeutics. Finally, we demonstrated the utility of our methods to sensitively determine in vivo viral titres directly from nasopharyngeal swabs overnight. When the latter values are combined with diagnostic PCR, this enabled real-time in vivo measures of infectivity to particle ratio calculations (viral fitness). Following characterization of our platform, Omicron arrived in Australia in late November of 2021. In this latter setting, we rapidly isolated and extensively characterized Omicron. This highlighted that the solutions provided herein were valuable for the continued COVID-19 response, as they enabled the development of a scale of relative variant threat that combined measures of viral fitness with measurements of humoral/therapeutic evasion to inform relevant national and international guidelines.

Results
Rapid isolation of all VOCs and major VUIs through Australian quarantine. In late 2020 and the first half of 2021, NSW hotel quarantine accommodated approximately half of all people entering Australia, with less than 1% being positive for SARS-CoV-2 infection (https://www.health.gov.au/sites/default/files/documents/2020/10/ national-review-of-hotel-quarantine.pdf). As part of the COVID-19 response, rapid molecular surveillance was established with 3rd generation single-molecule sequencing technologies 11 . In this setting, we introduced a hyper-permissive HEK239T-based cell line that co-expresses ACE2 and TMPRSS2 (HAT- 24), and was hyper-susceptible to rapid cytopathic effects (CPE) (Supplementary Fig. 1 and Video 1) compared with VeroE6-based cell lines. Susceptibility was based not only on the expression of physiologically relevant receptors ACE2 and TMPRSS2 15 , but also on the selection of a clone that has revealed -by substantial dose-dependent CPE accumulating after 8 h post infection (Supplementary Video 1) and after 3 d in culture -sensitivities in viral detection/isolation approaching that of diagnostic PCR (Supplementary Figs. 1 and 2). The HEK293T line has limited innate viral immunity [16][17][18][19][20] and whilst the latter lack of immunity may contribute to the phenotype, there was the only clone that was hyper-permissive to infection (see Supplementary Fig. 1a-f for a representative clone of HAT-24 (vs HAT-10) in 6 contemporary variants).
The introduction of the hyper-permissive line HAT-24 enabled virus isolation from greater than 80% of positive swabs, with all VOCs (Alpha, Beta, Gamma and Delta) and 6 VUIs (Kappa, Eta, Zeta, Epsilon, Iota and Lambda) isolated successfully over a four-month period in early 2021 (Fig. 1a). Visual scoring of cultures revealed that many were unequivocally positive for CPE after overnight culture and had extensive CPE after 48 h (Fig. 1b-d), with a significant correlation (r = −0.88; P = 0.02) between average diagnostic PCR Ct values and titres (TCID 50 ml −1 ) (Fig. 1a).
R-20 is a rapid, high-content assay for live SARS-CoV-2. Our next aim was to develop a fast, but low-cost, screening platform that could be scaled to high content. Using conventional approaches, we visually scored CPE to determine viral titres over 3 d to establish end-point titres as 50% Tissue Culture Infectious Dose (TCID 50 ) using the Spearman-Karber method 21 . In parallel, we used a high-content machine scoring method that enumerated the dose-dependent loss of 50% of nuclei (50% Lethal Infectious Dose (LD 50 )) through live cell staining with Hoechst-33342.
Machine-driven scoring and counting compared the VeroE6 and the HAT-24 cell lines in two formats: after 20 h and after 3 d of culture. Analysis of the 20 h truncated format was only performed using the HAT-24 line, as the VeroE6 derived cell lines could not succumb to sufficient CPE after 20 h (Fig. 2a). Enumeration of nuclei in HAT-24 line 20 h post infection through high-content imaging/analysis revealed dose-dependent loss of cells, with only minimal variation between replicates (Fig. 2c-e). While VeroE6 cultures also revealed  similar dose-dependent CPE, this occurred after 3 d and was accompanied by greater variability (Fig. 2b). End-point titres using the Spearman-Karber method were calculated at approximately 0.5 × 10 6 TCID 50 ml −1 in VeroE6 and 0.5 × 10 8 TCID 50 ml −1 in HAT-24 across all 12 variants tested (Fig. 2f,g). While we recognize the VeroE6 line to be relatively insensitive to infection, we further tested the more permissive VeroE6-TMPRSS2 cell line. The relative rank sensitivities of viral titres were HAT-24 > VeroE6-TMPRSS2 > VeroE6, with each increase in sensitivity being greater than an order of magnitude ( Supplementary Fig. 1g).
Given the linearity of the dose-dependent curves, for stringency and elimination of operator bias we established titres on the basis of 50% loss of nuclei (LD 50 ) 22 . This was then correlated with traditional TCID 50 assays after 3 d of culture using the Spearman-Karber method (Spearman coefficient of correlation, r = 0.62, P < 0.05) across all variant titres tested. We subsequently refer to the overnight method in HAT-24 as R-20, a rapid high-content 20 h full virus screening platform.
We then cross-validated the R-20 platform using VeroE6s 23 , given their availability and extensive use during the COVID-19 pandemic. For initial comparisons, we used the AB-3467 monoclonal antibody, as it is active against a broad range of SARS-CoV-2 variants 24 . We further cross-validated with the WHO international standard and reference panel for anti-SARS-CoV-2 antibody 25 , in addition to the Plasma Alliance (https://www.cslbehring.com/ newsroom/2020/covig19-plasma-alliance-expands-membership) internal serology standard. All cross-comparisons were carried out using 12 primary low-passage SARS-CoV-2 isolates. The R-20 platform observed all variants to be neutralized with similar IC 50 within the R-20 assay and in close correlation with that observed using VeroE6 (Fig. 2h-p). In addition, the R-20 platform revealed similar results to those laboratories that initially tested the WHO standard with live primary virus: 632 vs 686 for R-20 versus the mean contributing laboratories 25 . While R-20 is dependent on high-content microscopy and analysis, we further demonstrated that the R-20 assay is compatible with assays using standard optical multi-titre plate readers ( Supplementary Fig. 3a,b).

Immune evasion of SARS-CoV-2 at peak humoral responses.
For further stringent cross-validation, we turned to ranking of variants across a panel of sera from convalescent donors (n = 24) and mRNA vaccine (BNT162b2) recipients (n = 24). As observed with serological controls AB-3467 and WHO/Alliance standards, the R-20 platform produced similar results that were correlated with the longer yet more variable VeroE6 assays ( Fig. 3a-d).
Using both assays, we also observed differences across vaccinated and convalescent cohorts. For instance, across all vaccine recipients with titres greater than 1/160, 19 had neutralization breadth that was similar across all variants with the exception of Beta (Supplementary Tables 1, 3 and 5). In contrast, the panel of convalescent donors derived from the ADAPT cohort highlighted a continuum of different phenotypes (Supplementary Tables 2, 4 and  6.). For instance, two donors (AD010 and AD016; Supplementary  Fig. 4 and Table 2) maintained high titres and breadth across all variants, while one donor (AD062; Supplementary Fig. 4 and Table  2) had a potent response to early clade variants but could not reach end-point titres to the variants Epsilon, Delta, Kappa, Lambda, Gamma and Beta (Supplementary Table 2). With the exception of Gamma and Beta, all of these variants share the L452R or L452Q polymorphism in the Spike receptor-binding domain (RBD). In addition, while Zeta (D614G and E484K) was neutralized well across vaccine donors, it was significantly more evasive in convalescent responses (Fig. 3g-j). Overall, we observed Beta to be the most immune-evasive across both platforms, consistent with previous studies (Fig. 3g-j) 14,[26][27][28][29] .
In vivo infectivity and fitness of community-circulating variants. To establish the utility of the R-20 assay in determining viral infectivity from ex vivo samples, we curated two sets of specimens obtained during the acute phase of infection. In New South Wales, a quarantine breach at the end of 2020 led to singular seeding and community spread of the B.1.517.1 clade (Spike D614G and N501T). This clade provided primary samples with a genotype that was closely related to early circulating B clade D614G variant. The second cohort was derived during the month of July 2021 in the early stages of the Australian Delta outbreak. For each variant, samples were obtained at a time of no vaccination (B.1.517.1) or when 99% of reported cases (Delta) in NSW were transmissions in the unvaccinated (https://www.health.nsw.gov.au/Infectious/covid-19/ Documents/covid-surveillance-report-20210813.pdf). In addition, given the low prevalence of COVID-19 in Australia, the majority also represent their first infection. While we could rapidly identify those individuals with high infectious viral loads using R-20, we also used the longer culture period for increased sensitivity ( Fig.  4a-h). Using this strategy, we observed the sensitivity of 20 h vs 96 h cultures to be similar and their correlations with average Ct values to be similarly significant (r = -0.8585, P < 0.0001 and r = -0.8606, P < 0.0001, respectively). At both timepoints, virus could be detected at Ct values equal to and greater than 37 (Fig. 4h), thus approaching the sensitivity of diagnostic PCR assays.    Using linear regression ( Fig. 4i), we calculated Delta to be 9.1-fold more infectious than B.1.517.1, for samples with equivalent levels of viral RNA (P = 0.0004). There was no correlation with high viral loads and infectivity across age groups (r = 0.10556, P = 0.3674) (Fig. 4j). To conclude, this analysis confirmed that primary swabs can be used to calculate end-point titres and combined with diagnostic PCR values to rapidly determine relative viral fitness. The primary limitation to this latter fitness analysis was accumulation of primary swabs from the community. To address a scenario when this is limiting, we investigated the relative fitness of in vitro isolated and expanded Alpha, Delta and Lambda variants (as Delta has circulated and supplanted both Alpha and Lambda globally). As the closest surrogate for in vivo expansion, we infected primary air-liquid interface (ALI) cultures 30 with the same multiplicity of infection (MOI) using culture-expanded viral preparations. Replication in primary cultures would then reveal greater fitness of each isolate upon testing the titres in an identical manner to that used in patient nasopharyngeal swabs. Titres from primary ALI cultures 3 d post infection were observed to resolve the fitness of each variant, with Delta>Lambda>Alpha ( Fig. 5k), consistent with the known fitness within the community 31 .

Isolation and characterization of the first Australian Omicron case.
The first case of Omicron in Australia was detected on 27 November 2021 and upon receipt of the primary nasopharyngeal swab, virus was expanded in the HAT-24 line within 48 h (Fig. 5a) and then further expanded for 2 d using the VeroE6 cell line. Initial attempts to use the VeroE6-TMPRSS2 line were not successful, as limited to undetectable titres were observed after 24 to 48 h of culture. Given the rapid spread of Omicron in the community, we were able to access many primary nasopharyngeal swabs to test for infectivity per diagnostic Ct value. In testing 34 Omicron primary samples, we observed a significant downward shift in the linear regression, a finding in direct contrast to its known transmission fitness in the community (Fig. 5b). An initial hypothesis for this result is a change in the mechanism of viral entry compared with all previous SARS-CoV-2 variants studied herein. The downward shift in linear regression in the HAT-24 line and limited replication in the VeroE6-TMPRSS2 line, are two observations that support Omicron being less dependent on TMPRSS2. To test this hypothesis, we examined the ability of Omicron versus Delta to be blocked by the TMPRSS2 inhibitor Nafamostat using the HAT-24 line. In this setting, we observed Omicron to be refractory to Nafamostat at the highest concentration tested. Therefore, we  conclude that the cellular entry pathway for Omicron has diverged from all variants before Delta and throughout the pandemic. As TMPRSS2 significantly increases SARS-CoV-2 entry for other variants (especially Delta), the downward shift in the linear regression would be expected with Omicron using the HAT-24 cell line. Given the increased transmissibility of Omicron within the community, the use of other proteases and/or cathepsins that are present in the upper respiratory tract 32 may have resulted in this change in cellular tropism (Fig. 5d). Importantly, the R-20 platform revealed Omicron's lack of TMPRSS2 use and can readily resolve whether future SARS-CoV-2 variants either continue along this path in tropism or switch back to TMPRSS2-mediated entry. Importantly, at this juncture it must be noted that any measure of viral fitness in any assay will need to consider all pre-Omicron variants as TMPRSS2-dependent, and hence rank their fitness accordingly. In Omicron and related lineages that no longer or poorly utilize TMPRSS2, a similar grouping based on tropism will need to be applied before any comparisons of relative fitness are made.
Omicron resistance to humoral responses and immunotherapeutics. Neutralization of Omicron using sera from donors with two doses of BNT162b2 or ChAdOx1 nCoV-19 vaccines only reached end-point titres at 1/20 in 4 out of 17 donors tested (Supplementary Table 7). To determine titre reductions to statistical significance, we selected high neutralization titre serum samples from the Australian ADAPT cohort 23 (source of serum summarized in schematic from Supplementary Fig. 5). Neutralizations performed with convalescent sera from early clade infections (Fig. 6a,b and Supplementary Fig.  5a,b) showed an average of 4.7-fold reduction for Beta, 1.5-fold for Delta and 2.2-fold for Gamma relative to the ancestral strain (A.2.2). All responses with Omicron were below the limit of detection (serum dilution 1/20). For convalescent sera obtained from Delta wave infections ( Fig. 6c and Supplementary Fig. 5c), we observed a mean ID 50 of 52.6 for Omicron compared with mean ID 50 values of 770.5 for the ancestral strain, 211.1 for Beta, 317.5 for Gamma and 556.5 for Delta (Supplementary Table 8). Relative to the ancestral strain, this was a 20.7-fold reduction in neutralization with Omicron (P < 0.0001) compared with 4.0-fold for Beta (P < 0.0024), 1.6-fold for Delta and 2.9-fold for Gamma (P < 0.0365).
We next examined neutralization responses in sera from convalescent donors from early clade infections vaccinated with BNT162b2 or ChAdOx1 nCoV-19 vaccines (Fig. 6d,e and Supplementary Table 8). Here we again observe a 17.9 to 26.6-fold reduction in neutralization against Omicron (P < 0.0001) compared with 3.7 to 4.1-fold decrease observed for Beta (P < 0.0028). Similar results were observed with laboratory and healthcare worker volunteers at the peak of their third vaccine dose with BNT162b2, with a 16.9-fold reduction in neutralization against Omicron (P < 0.0001) compared with a 4.4-fold reduction for Beta (P < 0.0001) ( Fig. 6f and Supplementary Table 9).
To observe neutralization responses at the population level, we then tested five polyclonal human IgG batches which comprise more than 10,000 pooled plasma donors collected during the peak of the US vaccine rollout ( Fig. 6g and Supplementary Table 8). There was a 16.8-fold reduction in neutralization against Omicron (P < 0.0001) compared with a 3.3-fold decrease for Beta (P = 0.0437). Similar fold reductions were also observed from polyclonal IgG that was collected from convalescent donors between September and October 2020 (fold reduction of 14.1-fold for Omicron; P < 0.0001 versus 3.5-fold for Beta; P < 0.05) (Fig. 6g). Using the highest titre IgG batch (Poly_IgG -1033) and one based solely on convalescent donors (Con-Poly_IgG -869), we then tested all variants against Omicron to establish a ranking of humoral evasion to all dominant variants circulating in 2020 to 2021 (Fig. 6i,j).
We then determined activity across clinically available monoclonal antibodies (mAb) presently used in therapy. The class 3 antibody Sotrovimab, which targets a highly conserved region of the sarbecovirus RBD 33 , retained neutralizing activity against Omicron. For other monoclonal antibodies, we observed either a lack of neutralization (Casirivimab, Imdevimab and Bamlaninvimab) or significant reduction thereof (Tixagevimab) ( Table 1). These data were cited by a US Centers for Disease Control and Prevention (CDC) Health Advisory, forming updated recommendations for the use of monoclonal antibodies to treat COVID-19 when the Omicron BA1 lineage was accelerating in spread (https://emergency.cdc.gov/ han/2021/han00461.asp.).
Omicron and implications for vaccine effectiveness. We next estimated the fold reduction in neutralization for each variant within each cohort (with censoring). To do so, we grouped the data into three convalescent groups (Conv1-first wave, Conv2-second wave, Conv3-third wave (VOC Delta); see A, B and C in Supplementary  Fig. 5), convalescent plus vaccinated, vaccine boosted (third dose), pooled polyclonal IgG and the WHO international reference standard as a control. The data are summarized in Fig. 6h. We have previously demonstrated that the neutralizing antibody titre is highly predictive of protection from symptomatic SARS-CoV-2 infection 34 , and that the drop in titre to a given variant can be used to predict protection against the variant 35 . Using the protection curve in Khoury et al. 34 , we estimated the level of protection for BNT162b2-vaccinated or boosted individuals in the first few months after vaccination (Table 2). This predicts a substantial drop in protection from symptomatic infection for BNT162b2-vaccinated and boosted individuals (compared with ancestral virus) 36 , although with a relative preservation of protection from severe infection.

Discussion
Our aim was to enable platforms to accelerate the COVID-19 response at two principal levels. First, the rapid and sensitive isolation of primary SARS-CoV-2 isolates down to low viral loads. Second, enabling rapid characterization of their relative threat. The latter ranged from the ability to rapidly determine the evasion potential against a humoral response and/or therapeutics through to assessment of viral fitness in vivo and in vitro. For rapidly spreading pandemics such as COVID-19, the time taken for stringent resolution of a viral phenotype often lags. Time and precision are paramount in this context and the R-20 platform enables this at several levels, from time to virus isolation and expansion through to characterization.
Variants before Omicron all share similar changes within their Spike glycoprotein [1][2][3][4] . The changes are related to humoral evasion and viral fitness gains that have significantly increased transmissibility in variants such as Delta. While Beta was initially the most evasive variant in early 2021, Omicron emerged in late November as F G Delta the most evasive and transmissible variant observed. Using the R-20 platform, we rapidly isolated then ranked Omicron across a continuum of humoral immunity in the community. Simultaneous to the submission of this work for review, many laboratories published pre-prints on the relative fold reduction of neutralization titres of Omicron [37][38][39] . As observed in the study of previous variants, the fold reductions covered a significant range. Importantly, it is imperative that we align actual vaccine efficacy with what we observe in vitro. Using data observed herein and an immunobridging modelling approach, we calculate a reduction in two-dose vaccine effectiveness to 37.2%, within the range of actual vaccine effectiveness observed in South Africa at 33% (https://www.discovery.co.za/corporate/ health-insights-omicron-outbreak-analysis). While Omicron represented a significant challenge to the existing two-dose vaccination strategy, it fortunately has not translated to the mortality observed in unvaccinated communities in the early phases of the pandemic 40 . Infection in vaccinated populations is consistent with Omicron's ability to evade humoral immunity. Disease severity following infection is multifactorial and would be tempered by both humoral and cellular immunity acquired through vaccination and/or previous infection. Yet not everyone in the community, such as the elderly or the immunocompromised, can establish a vaccine response that can lead to protection from severe disease. Fortunately for these at-risk groups, immunotherapeutic treatments such as Sotrovimab retain neutralizing activity for the Omicron lineage BA1. Unfortunately, preliminary data on the Omicron BA2 lineage have observed greater resistance to Sotrovimab (https://doi.org/10.1056/NEJMc2201933) and moving forward, therapeutics not targeting Spike, such as the protease inhibitor Paxlovid, may be more pragmatic for the treatment of at-risk groups against future circulating variants. Viral loads based on diagnostic PCR with a variant in either unvaccinated or breakthrough infection [41][42][43] cannot often inform on the real risk of an individual's ability to spread a particular circulating variant or how previous vaccination may influence this. Furthermore, many people may be positive from diagnostic PCR but represent limited risk in spreading the virus in the community. Through rapid end-point titres on the R-20 platform, we demonstrated infectious potential in an individual, and the relative infectivity per viral load between variants. This work also highlighted the inefficient use of TMPRSS2 and the divergence of the Omicron variant to another entry pathway. While preliminary studies support Omicron's cellular entry to be preferentially by endocytosis 32 , an alternative entry pathway using ACE2 and a co-factor (for example protease) enriched in bronchial tissue would also be consistent with not only the observed shift in its tropism, but also the increase in its transmissibility. Importantly, if lower disease severity is linked to higher bronchial infection as observed in animal models 44,45 , it is imperative that we continue to rapidly observe what entry pathways are used by emerging variants. If variants appear that again primarily use the ACE2-TMPRSS2 pathway, the platform herein can be readily used to resolve their appearance. This is similar to what was previously observed in other viral families, with the most documented being that for HIV-1 and its ability to chemokine receptor switch between CCR5 and CXCR4 46 .
To conclude, the research herein provides an accessible, easy to use pipeline that enables resolution of SARS-CoV-2 variant threat at several important levels. First, in its ability to sensitively combine viral isolation, expansion and characterization of primary viral isolates from nasopharyngeal samples within one week. Second, in its ability to enable screening of thousands of compounds/samples reproducibly, efficiently, at low cost and at scale. While this was used in the resolution of the key phenotypes of many contemporary SARS-CoV-2 variants that emerged in 2021, its utility was tested with the emergence of Omicron at the conclusion of this study.

Methods
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 guideline 47 . 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). 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 Australia 23 , 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 virus 48 .

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 confirmation 49 , the immunoglobulin was purified using the licensed and fully validated immunoglobulin manufacturing process used for Privigen 50 , notionally similar to others 51 . 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 NIBSC 25 .

Generation of HEK293T-ACE2-TMPRSS2 cells (clone HAT-24).
HEK293T cells stably expressing human ACE2 and TMPRSS2 were generated by lentiviral transductions as previously described 23 . Briefly, the open reading frames for hACE2 (Addgene, 1786) and hTMPRSS2a (IDT, synthetic gene fragment) were cloned into lentiviral expression vectors pRRLsinPPT.CMV.GFP.WPRE 52 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 *Vaccine effectiveness against severe outcomes is much less well-validated as there is insufficient data available with which to parameterize the model at such low titres against severe outcomes, in addition to a lack of understanding of baseline severity with Omicron. 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 described 53 . 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 experiments 23 .
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 10 4 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 × 10 6 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 × 10 7 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 × 10 4 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 described 11 . Consensus SARS-CoV-2 genomes have been uploaded to GISAID (www.gisaid.org) and are publicly available as indicated in Supplementary Table 1. 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 by two independent experienced operators and compared against negative and positive infection controls to score wells binarily for CPE (+ or −) to calculate TCID 50 values according to the Spearman-Karber method 21 . Fluorescence images were processed with IN Carta analysis software (Cytiva) to obtain total nuclei counts per well. For calculation of LD 50 values, cell counts were normalized so that 100% represents the average cell number for mock-infected controls and 0% represents the average cell number for the highest viral concentration tested. LD 50 values were obtained with GraphPad Prism software using the nonlinear regression for dose-response with variable slope and four parameters. Viral titration on VeroE6 cells was performed exactly as described above, except that cells were seeded at 5 × 10 3 cells per well in MEM-2% FBS medium and stained with Hoechst-33342 after a 72 h incubation.
Rapid high-content SARS-CoV-2 microneutralization assay with HAT-24 cells (overnight R-20 assay). 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 × 10 4 cells per well. Human sera or monoclonal antibodies were serially diluted (2-fold) in DMEM-5% FBS and mixed in duplicate with an equal volume of SARS-CoV-2 virus solution at 2× the median lethal dose (2 × LD 50 ), calculated as indicated above. After 1 h of virus-serum co-incubation at 37 °C, 40 μl was added to an equal volume of pre-plated cells. Cell plates were then incubated for 20 h before direct imaging on an InCell Analyzer HS2500 high-content fluorescence microscopy system (Cytiva). Cellular nuclei counts were obtained with IN Carta automated image analysis software (Cytiva), and the percentage of virus neutralization was calculated with the formula: %N = (D−(1−Q)) × 100/D, where 'Q' is a well's nuclei count divided by the average count for uninfected controls (defined as having 100% neutralization) and D = 1−Q for the average count of positive infection controls (defined as having 0% neutralization). For additional detail and the rationale behind this formula, see Supplementary Fig. 8 in ref. 23 . The cut-off for determining the neutralization end-point titre of diluted serum samples was set to the last consecutive dilution reaching ≥50% neutralization for the average of technical replicates. Unless otherwise specified, samples were tested at a starting dilution of 1/20.  Estimating vaccine effectiveness against Omicron. Vaccine effectiveness (VE) against Omicron was estimated using the approach and model previously reported 34,35 . Briefly, we estimated VE for a given vaccine/cohort against a given variant on the basis of the (log 10 ) geometric mean (GM) neutralization titre, normalized to the GM neutralization titre (against ancestral virus) of convalescent individuals (exposed to ancestral virus). This normalized (log 10 ) GM titre is given by μ, and the VE for a given μ is predicted using the equation

High-content SARS-CoV
where N is the probability density function of a normal distribution of the distribution in log 10 neutralization titres with mean μ and standard deviation σ, and x 50 and k are the parameters of a logistic function representing the log 10 -normalized neutralization titre associated with 50% protection and k determines the steepness of the logistic function. The parameters σ = 0.46 (representing the spread of neutralization titres in vaccinated individuals), x 50 = log 10 (0.20) and k = 3.1 were estimated previously by fitting vaccine efficacy data from randomized control trials across 7 SARS-CoV-2 vaccines 34 . Thus, vaccine efficacy of BNT162b2-vaccinated individuals against Omicron is estimated by the normalized GM neutralization titre of BNT162b2-vaccinated individuals against Omicron. Previously, we have estimated that BNT162b2-vaccinated individuals have a GM neutralization titre (against ancestral virus) that is 2.4-fold the GM neutralization titre of convalescent individuals (who were exposed to ancestral virus) 34 . Thus, to estimate the VE against Omicron of BNT162b2-vaccinated individuals, we calculated the normalized GM neutralization titre of these individuals against Omicron. A 20.2-fold drop in neutralization against Omicron was estimated to lead to a GM neutralization titre in BNT162b2-vaccinated individuals against Omicron of 2.4/20.2 = 0.119-fold of the GM neutralization titre of convalescent individuals (after exposure to ancestral virus) against ancestral virus (thus, μ = log 10 (0.119) in the above equation to estimate vaccine efficacy for BNT162b2-vaccinated individuals against Omicron). Similarly, we have previously shown that mRNA vaccines in previously infected individuals produced GM neutralization titres that are approximately 12.0-fold higher than the GM titres of convalescent individuals who were exposed to ancestral virus 54 . Thus, the 20.2-fold loss of neutralization to Omicron was estimated to give a GM neutralization titre against Omicron that is 0.594-fold of the GM titre of convalescent participants against ancestral virus (after exposure to ancestral virus). Thus, to estimate vaccine efficacy for previously infected and mRNA-vaccinated individuals, we used μ = log 10 (0.594) in the above equation. Confidence intervals for predicted vaccine efficacies were estimated using bootstrapping.
Materials availability. This study generated a new reagent described as HEK293T-ACE2-TMPRSS2-Clone#24 (HAT-24) cell line. HAT-24 cells will be made available through CellBank Australia, ATCC and ECACC. Laboratories unable to source this cell line (or until this cell line is available at these collections) from the latter organizations can directly obtain the cell line through the corresponding author through a standard material transfer agreement.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
All viral sequences have been deposited with the NIH BioProject under Project #PRJNA824540. GISAID reference numbers are listed in Supplementary

Code availability
No custom code was generated or used.

March 2021
Corresponding author(s): Stuart Turville Last updated by author(s): Apr 7, 2022 Reporting Summary Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our Editorial Policies and the Editorial Policy Checklist.

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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Whole-genome viral sequences for the SARS-CoV-2 variants used in this study have been deposited to GISAID and are publicly available as of the date of publication. Accession numbers are listed in