Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization

The emergence of the SARS-CoV-2 variant of concern Omicron (Pango lineage B.1.1.529), first identified in Botswana and South Africa, may compromise vaccine effectiveness and lead to re-infections1. Here we investigated Omicron escape from neutralization by antibodies from South African individuals vaccinated with Pfizer BNT162b2. We used blood samples taken soon after vaccination from individuals who were vaccinated and previously infected with SARS-CoV-2 or vaccinated with no evidence of previous infection. We isolated and sequence-confirmed live Omicron virus from an infected person and observed that Omicron requires the angiotensin-converting enzyme 2 (ACE2) receptor to infect cells. We compared plasma neutralization of Omicron relative to an ancestral SARS-CoV-2 strain and found that neutralization of ancestral virus was much higher in infected and vaccinated individuals compared with the vaccinated-only participants. However, both groups showed a 22-fold reduction in vaccine-elicited neutralization by the Omicron variant. Participants who were vaccinated and had previously been infected exhibited residual neutralization of Omicron similar to the level of neutralization of the ancestral virus observed in the vaccination-only group. These data support the notion that reasonable protection against Omicron may be maintained using vaccination approaches.

The emergence of the SARS-CoV-2 variant of concern Omicron (Pango lineage B.1.1.529), first identified in Botswana and South Africa, may compromise vaccine effectiveness and lead to re-infections 1 . Here we investigated Omicron escape from neutralization by antibodies from South African individuals vaccinated with Pfizer BNT162b2. We used blood samples taken soon after vaccination from individuals who were vaccinated and previously infected with SARS-CoV-2 or vaccinated with no evidence of previous infection. We isolated and sequence-confirmed live Omicron virus from an infected person and observed that Omicron requires the angiotensin-converting enzyme 2 (ACE2) receptor to infect cells. We compared plasma neutralization of Omicron relative to an ancestral SARS-CoV-2 strain and found that neutralization of ancestral virus was much higher in infected and vaccinated individuals compared with the vaccinated-only participants. However, both groups showed a 22-fold reduction in vaccine-elicited neutralization by the Omicron variant. Participants who were vaccinated and had previously been infected exhibited residual neutralization of Omicron similar to the level of neutralization of the ancestral virus observed in the vaccination-only group. These data support the notion that reasonable protection against Omicron may be maintained using vaccination approaches.
The emergence of the Omicron variant of SARS-CoV-2 in November 2021, first identified in South Africa and Botswana, was first described in South Africa 2 , followed shortly afterwards by confirmed transmission in Hong Kong 3 . Owing to the large number of mutations in the spike protein and elsewhere on the virus (https://covdb.stanford.edu/page/ mutation-viewer/#omicron), there is concern that this variant will exhibit substantial escape from vaccine-elicited immunity 4,5 . Furthermore, several mutations in the spike receptor-binding domain and S2 fusion domain are predicted to increase transmission 5 .
Here we have used the human lung cell line H1299-ACE2 (Extended Data Fig. 1), which overexpresses the human ACE2 receptor 6 , to both isolate Omicron and test its neutralization by human plasma. We isolated Omicron virus using one passage on H1299-ACE2 cells and a second passage on H1299-ACE2 cells in co-culture with the Vero E6 African green monkey kidney cell line. Sequencing of the isolated virus confirmed it was the Omicron variant bearing the R346K mutation. We observed no mutations introduced in vitro as majority or minority variants (Extended Data Table 1). H1299-ACE2 cells were similar to Vero E6 cells in that they formed infection foci during infection with ancestral D614G and Beta variant viruses; however, the H1299-ACE2 cells formed more foci than unmodified Vero E6 cells (Extended Data Fig. 2a, b). Infection by cell-free Omicron of unmodified Vero E6 cells was inefficient (Extended Data Fig. 2c) and we could not use cell-free Omicron infection in Vero E6 cells to generate a useable virus stock of this isolate (Extended Data Fig. 2d).
We observed that Omicron infected the H1299-ACE2 cells in a concentration-dependent manner but did not infect the parental H1299 Nature | Vol 602 | 24 February 2022 | 655 cells, indicating that human ACE2 is required for Omicron entry (Fig. 1a, b). We then tested the ability of plasma from individuals vaccinated with BNT162b2 to neutralize Omicron versus ancestral D614G virus in a live virus neutralization assay. We tested plasma samples taken from 19 individuals after they had received 2 doses of BNT162b2 (Extended Data Tables 2, 3), 6 of whom had no previous record of SARS-CoV-2 infection or detectable SARS-CoV-2 nucleocapsid antibodies indicative of previous infection (Methods). We also tested samples from a later time point for two of the vaccinated-only participants (Extended Data Table 3). The previously infected and vaccinated participants were infected with either ancestral SARS-CoV-2 strains or the Delta variant (Extended Data Table 3). To quantify neutralization in the live virus neutralization assay, we calculated the focus reduction neutralization test value (FRNT 50 , the inverse of the plasma dilution required for a 50% reduction in infection focus number).
We compared these results with neutralization of the Beta variant 6,10-16 using Beta and D614G virus infection of H1299-ACE2 (Extended Data Fig. 3a) and Vero E6 (Extended Data Fig. 3b) cells. The fold difference relative to the ancestral D614G virus was 4.3 for H1299-ACE2 cells and 5.0 for Vero E6 cells. Thus, results from these two cell lines indicated that Omicron exhibited approximately fourfold greater escape relative to Beta in our assays.
This study was not designed to reliably evaluate vaccine efficacy or protection from severe disease. However, a prediction of vaccine efficacy after a 22-fold drop in neutralization can be made in individuals vaccinated with BNT162b2 and individuals who were both vaccinated and boosted on the basis of data from randomized control trials using a model relating neutralization level to vaccine efficacy 17,18 . Using this model and the differences in neutralization between Omicron and other SARS-CoV-2 strains (Methods), we predict a vaccine efficacy for preventing symptomatic infection by Omicron of 73% (95% confidence interval 58-83%) for vaccinated and boosted individuals, and 35% (95% confidence interval 20-50%) for vaccinated-only individuals; this suggests that Omicron compromises the ability of the vaccine to protect against infection in individuals in the vaccinated-only group but not in vaccinated and boosted individuals (Fig. 1e). We note that these predictions are similar to reports of actual vaccine efficacy in the UK 19 .
Shortly after we released these findings, several other groups have reported similar results 3,20-23 including Pfizer-BioNTech (https://www. businesswire.com/news/home/20211208005542/en/). These results mirror ours, with large reductions in neutralization of Omicron compared with ancestral virus by vaccine-elicited immunity, neutralizing monoclonal antibodies and plasma from convalescent individuals previously infected with other variants. Notably, the Pfizer-BioNTech study reports that boosting seems to increase neutralization breadth, which reduces the escape by Omicron relative to ancestral virus; these results have been validated independently 21 . Unlike what was reported for boosting, we did not observe a lower fold drop in previously vaccinated and infected relative to the vaccinated-only participants in this study.
Limitations of this study include the presence of an R346K substitution in our virus stock. This putative escape mutation 24 , which may confer moderate antibody resistance (https://jbloomlab.github.io/ SARS2_RBD_Ab_escape_maps/escape-calc/), is not found in the majority Twenty-one samples were tested from n = 19 participants in 2 independent experiments (n = 13 vaccinated and previously infected; n = 6 vaccinated only). Grey points denote measurements where 50% neutralization was not achieved with the most concentrated plasma used. P = 4.8 × 10 −5 , Wilcoxon rank-sum test. d, Geometric mean and 95% confidence interval of the fold change in neutralization between ancestral D614G and Omicron neutralization in plasma. Purple denotes all participants, blue denotes vaccinated individuals with previous SARS-CoV-2 infection, orange denotes vaccinated-only individuals, and yellow denotes all participants excluding those in whom 50% neutralization was not achieved. e, Mean predicted vaccine efficacy and 95% confidence intervals against symptomatic infection with Omicron using data from previous randomized controlled trials and the 22-fold difference between D614G and Omicron observed in this study 17,18 . Predictions are for vaccinated and boosted (B, red) or vaccinated-only (V, blue) individuals.
Article of Omicron genomes. In addition, the timing of sample collection soon after vaccination (Supplementary Tables 2, 3) does not account for the waning of neutralization capacity 25,26 .
So far, a milder course of Omicron infection has been observed in South Africa relative to previous infection waves in terms of reported numbers of patients in intensive care units and needing ventilation 27 . Although there may be other unidentified contributing factors that lower pathogenicity 28 , pre-existing immunity would be expected to be higher in the Omicron wave because of vaccination as well as immunity elicited by previous infection during one of three preceding infection waves in South Africa 28 . Therefore, the incomplete Omicron escape from previous immunity described here may be an important factor accounting for the milder course of infection. Despite the extensive neutralization escape of Omicron, residual neutralization levels may still be sufficient to protect from severe disease 17,18 . Other facets of the adaptive immune response elicited by vaccination and previous infection may increase protection. Furthermore, we observed that vaccination combined with previous infection elicits similar neutralization capacity against Omicron as vaccination without previous infection elicits against ancestral virus. This indicates that protection from symptomatic Omicron infection may occur when vaccination is combined with previous infection or boosting. This may explain why Pfizer BNT162b2 vaccination has been shown to substantially decrease the risk of hospital admission caused by Omicron infection in South Africa 29 and supports the use of further vaccination and boosting to combat Omicron.

Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-021-04387-1.

Methods
Whole-genome sequencing, genome assembly and phylogenetic analysis cDNA synthesis was performed on the extracted RNA using random primers followed by gene-specific multiplex PCR using the ARTIC V.3 protocol (https://www.protocols.io/view/covid-19-artic-v3-ill umina-library-construction-an-bibtkann). In brief, extracted RNA was converted to cDNA using the Superscript IV First Strand synthesis system (Life Technologies) and random hexamer primers. SARS-CoV-2 whole-genome amplification was performed by multiplex PCR using primers designed using Primal Scheme (http://primal.zibraproject. org/) to generate 400-bp amplicons with an overlap of 70 bp that covers the 30-kb SARS-CoV-2 genome. PCR products were cleaned up using AmpureXP purification beads (Beckman Coulter) and quantified using the Qubit dsDNA High Sensitivity assay on the Qubit 4.0 instrument (Life Technologies). We then used the Illumina Nextera Flex DNA Library Prep kit according to the manufacturer's protocol to prepare indexed paired-end libraries of genomic DNA. Sequencing libraries were normalized to 4 nM, pooled and denatured with 0.2 N sodium acetate. Then, a 12-pM sample library was spiked with 1% PhiX (a PhiX Control v.3 adaptor-ligated library was used as a control). We sequenced libraries on a 500-cycle v.2 MiSeq Reagent Kit on the Illumina MiSeq instrument (Illumina). We assembled paired-end fastq reads using Genome Detective 1.126 (https://www.genomedetective.com) and the Coronavirus Typing Tool. We polished the initial assembly obtained from Genome Detective by aligning mapped reads to the reference sequences and filtering out low-quality mutations using the bcftools 1.7-2 mpileup method. Mutations were confirmed visually with BAM files using Geneious software (Biomatters).

SARS-CoV-2 nucleocapsid enzyme-linked immunosorbent assay (ELISA)
Nucleocapsid protein (2 μg ml −1 ) (Biotech Africa; catalogue (cat.) no. BA25-P) was used to coat 96-well, high-binding plates and incubated overnight at 4 °C. The plates were incubated in a blocking buffer consisting of 5% skimmed milk powder, 0.05% Tween 20, 1× PBS. Plasma samples were diluted to a 1:100 dilution in a blocking buffer and added to the plates. Horseradish peroxidase (HRP)-conjugated IgG secondary antibody was diluted to 1:3,000 in blocking buffer and added to the plates followed by tetramethylbenzidine (TMB) peroxidase substrate (Thermo Fisher Scientific). Upon stopping the reaction with 1 M H 2 SO 4 , absorbance was measured at a 450-nm wavelength.

Cells
Vero E6 cells (ATCC CRL-1586, obtained from Cellonex) were propagated in complete growth medium consisting of Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (Hyclone) containing 10 mM of HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine and 0.1 mM nonessential amino acids (Sigma-Aldrich). Vero E6 cells were passaged every 3-4 days. H1299 cell lines were propagated in growth medium consisting of complete Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum containing 10 mM of HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine and 0.1 mM nonessential amino acids. H1299 cells were passaged every second day. The H1299-E3 (H1299-ACE2, clone E3) cell line was derived from H1299 (CRL-5803) as described in previous work 6 and Supplementary Fig. 1. In brief, vesicular stomatitis virus G glycoprotein (VSVG) pseudotyped lentivirus containing ACE2 was used to spinfect H1299 cells. ACE-2 transduced H1299 cells (containing an endogenously yellow fluorescent protein labelled histone H2AZ gene 30 ) were then subcloned at single-cell density in 96-well plates (Eppendorf) in conditioned medium derived from confluent cells. After 3 weeks, wells were detached using a 0.25% trypsin-EDTA solution (Gibco) and plated in 2 replicate plates, where the first plate was used to determine infectivity and the second was stock. The first plate was screened for the fraction of mCherry-positive cells per cell clone upon infection with a SARS-CoV-2 mCherry expressing spike pseudotyped lentiviral vector. Screening was performed using a Metamorph-controlled (Molecular Devices) Nikon TiE motorized microscope (Nikon) with a 20×, 0.75 NA phase objective, 561-nm laser line, and 607-nm emission filter (Semrock). Images were captured using an 888 EMCCD camera (Andor). The clone with the highest fraction of mCherry expression was expanded from the stock plate and denoted H1299-E3. Infectivity was confirmed with mCherry expressing lentivirus by flow cytometry using a BD Fortessa instrument and analysed using BD FACSDiva Software (BD Biosciences). This clone was used in the outgrowth and focus forming assay. Cell lines have not been authenticated. The cell lines have been tested for mycoplasma contamination and are mycoplasma negative.

Virus expansion
All work with live virus was performed in biosafety level 3 containment using protocols for SARS-CoV-2 approved by the Africa Health Research Institute Biosafety Committee. ACE2-expressing H1299-E3 cells were seeded at 4.5 × 10 5 cells in a well on a 6-well plate and incubated for 18-20 h. After one DPBS wash, the sub-confluent cell monolayer was inoculated with 500 μl universal transport medium diluted 1:1 with growth medium filtered through a 0.45-μm filter. Cells were incubated for 1 h. Wells were then filled with 3 ml complete growth medium. After 4 days of infection (completion of passage 1 (P1)), cells were trypsinized, centrifuged at 300g for 3 min and resuspended in 4 ml growth medium. Then, 2 ml was added to Vero E6 cells that had been seeded at 2 × 10 5 cells per ml, 5 ml total, 18-20 h earlier in a T25 flask (approximately 1:8 donor-to-target cell dilution ratio) for cell-to-cell infection. The co-culture of ACE2-expressing H1299-E3 and Vero E6 cells was incubated for 1 h and 7 ml of complete growth medium was added to the flask and incubated for 4 days. The viral supernatant (passage 2 (P2) stock) was used for experiments. Further optimization of the viral outgrowth protocol used for subsequent Omicron isolates showed that addition of 4 ml instead of 2 ml of infected H1299-E3 cells to Vero E6 cells that had been seeded at 2 × 10 5 cells per ml, 20 ml total, 18-20 h earlier in a T75 flask gave P2 stocks with substantially higher titres that could detectably infect Vero E6 cells. The Omicron virus isolate is available from the authors contingent on verification that it will be received and used in a biosafety level 3 facility.
Live virus neutralization assay H1299-E3 cells were plated in a 96-well plate (Corning) at 30,000 cells per well 1 day before infection. Plasma was separated from EDTA-anticoagulated blood by centrifugation at 500g for 10 min and stored at −80 °C. Aliquots of plasma samples were heat-inactivated at 56 °C for 30 min and clarified by centrifugation at 10,000g for 5 min. Virus stocks were used at approximately 50-100 focus-forming units per microwell and added to diluted plasma. Antibody-virus mixtures were incubated for 1 h at 37 °C, 5% CO 2 . Cells were infected with 100 μl of the virus-antibody mixtures for 1 h, then 100 μl of a 1× RPMI 1640 (Sigma-Aldrich, R6504), 1.5% carboxymethylcellulose (Sigma-Aldrich, C4888) overlay was added without removing the inoculum. Cells were  Table S2 and S3, where n = 6 were vaccinated only and n = 13 were vaccinated and previously infected. p = 0.006 for both (A) and (B) as determined by the Wilcoxon rank sum test.