Abstract
The Omicron (B.1.1.529) variant of SARS-CoV-2 was only recently detected in southern Africa, but its subsequent spread has been extensive, both regionally and globally1. It is expected to become dominant in the coming weeks2, probably due to enhanced transmissibility. A striking feature of this variant is the large number of spike mutations3 that pose a threat to the efficacy of current COVID-19 vaccines and antibody therapies4. This concern is amplified by the findings from our study. We found B.1.1.529 to be markedly resistant to neutralization by serum not only from convalescent patients, but also from individuals vaccinated with one of the four widely used COVID-19 vaccines. Even serum from persons vaccinated and boosted with mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies to all known epitope clusters on the spike protein, we noted that the activity of 18 of the 19 antibodies tested were either abolished or impaired, including ones currently authorized or approved for use in patients. In addition, we also identified four new spike mutations (S371L, N440K, G446S, and Q493R) that confer greater antibody resistance to B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2.
Main text
The COVID-19 pandemic rages on, as the causative agent, SARS-CoV-2, continues to evolve. Many diverse viral variants have emerged (Fig. 1a), each characterized by mutations in the spike protein that raise concerns of both antibody evasion and enhanced transmission. The Beta (B.1.351) variant was found to be most refractory to antibody neutralization4 and thus compromised the efficacy of vaccines5-7 and therapeutic antibodies. The Alpha (B.1.1.7) variant became dominant globally in early 2021 due to an edge in transmission8 only to be replaced by the Delta (B.1.617.2) variant, which exhibited even greater propensity to spread coupled with a moderate level of antibody resistance9. Then came the Omicron (B.1.1.529) variant, first detected in southern Africa in November 20213,10,11 (Fig. 1a). It has since spread rapidly in the region, as well as to over 60 countries, gaining traction even where the Delta variant is prevalent. The short doubling time (2-3 days) of Omicron cases suggests it could become dominant soon2. Moreover, its spike protein contains an alarming number of >30 mutations (Fig. 1b and Extended Data Fig. 1), including at least 15 in the receptor-binding domain (RBD), the principal target for neutralizing antibodies. These extensive spike mutations raise the specter that current vaccines and therapeutic antibodies would be greatly compromised. This concern is amplified by the findings we now report.
a, Unrooted phylogenetic tree of B.1.1.529 with other major SARS-CoV-2 variants. b, Key spike mutations found in the viruses isolated in the major lineage of B.1.1.529 are denoted. c, Neutralization of D614G and B.1.1.529 pseudoviruses by convalescent patient sera. d, Neutralization of D614G and B.1.1.529 pseudoviruses by vaccinee sera. Within the four standard vaccination groups, individuals that were vaccinated without documented infection are denoted as circles and individuals that were both vaccinated and infected are denoted as triangles. Within the boosted group, Moderna vaccinees are denoted as squares and Pfizer vaccinees are denoted as diamonds. e, Neutralization of authentic D614G and B.1.1.529 viruses by vaccinee sera. Moderna vaccinees are denoted as squares and Pfizer vaccinees are denoted as diamonds. For all panels, values above the symbols denote geometric mean titer and the numbers in parentheses denote the sample size. P values were determined by using a Wilcoxon matched-pairs signed-rank test (two-tailed).
Serum neutralization of B.1.1.529
We first examined the neutralizing activity of serum collected in the Spring of 2020 from COVID-19 patients, who were presumably infected with the wild-type SARS-CoV-2 (see Methods). Samples from 10 individuals were tested for neutralization against both D614G (WT) and B.1.1.529 pseudoviruses. While robust titers were observed against D614G, a significant drop (>32-fold) in ID50 titers was observed against B.1.1.529, with only 2 samples showing titers above the limit of detection (LOD) (Fig. 1c and Extended Data Fig. 2a). We then assessed the neutralizing activity of sera from individuals who received one of the four widely used COVID-19 vaccines: BNT162b2 (Pfizer), mRNA-1273 (Moderna), Ad26.COV2.S (Johnson & Johnson), and ChAdOx1 nCoV-19 (AstraZeneca) (see Methods and Extended Data Table 1). In all cases, a substantial loss in neutralizing potency was observed against B.1.1.529 (Fig. 1d and Extended Data Fig. 2b-f). For the two mRNA-based vaccines, BNT162b2 and mRNA-1273, a >21-fold and >8.6-fold decrease in ID50 was seen, respectively. We note that, for these two groups, we specifically chose samples with high titers such that the fold-change in titer could be better quantified, so the difference in the number of samples having titers above the LOD (6/13 for BNT162b2 versus 11/12 for mRNA-1273) may be favorably biased. Within the Ad26.COV2.S and ChAdOx1 nCOV-19 groups, all samples were below the LOD against B.1.1.529, except for two Ad26.COV2.S samples from patients with a previous history of SARS-CoV-2 infection (Fig. 1d). Collectively, these results suggest that individuals who were previously infected or fully vaccinated remain at risk for B.1.1.529 infection.
Booster shots are now routinely administered in many countries 6 months after full vaccination. Therefore, we also examined the serum neutralizing activity of individuals who had received three homologous mRNA vaccinations (13 with BNT162b2 and 2 with mRNA-1273). Every sample showed lower activity in neutralizing B.1.1.529, with a mean drop of 6.5-fold compared to WT (Fig. 1d). Although all samples had titers above the LOD, the substantial loss in activity may still pose a risk for B.1.1.529 infection despite the booster vaccination.
We then confirmed the above findings by testing a subset of the BNT162b2 and mRNA-1273 vaccinee serum samples using authentic SARS-CoV-2 isolates: wild type and B.1.1.529. Again, a substantial decrease in neutralization of B.1.1.529 was observed, with mean drops of >6.0-fold and >4.1-fold for the fully vaccinated group and the boosted group, respectively (Fig. 1e).
Monoclonal antibody neutralization of B.1.1.529
To understand the types of antibodies in serum that lost neutralizing activity against B.1.1.529, we assessed the neutralization profile of 19 well-characterized monoclonal antibodies (mAbs) to the spike protein, including 17 directed to RBD and 2 directed to the N-terminal domain (NTD). We included mAbs that have been authorized or approved for clinical use, either individually or in combination: REGN10987 (imdevimab)12, REGN10933 (casirivimab)12, COV2-2196 (tixagevimab)13, COV2-2130 (cilgavimab)13, LY-CoV555 (bamlanivimab)14, CB6 (etesevimab)15, Brii-196 (amubarvimab)16, Brii-198 (romlusevimab)16, and S309 (sotrovimab)17. We also included other mAbs of interest: 910-3018, ADG-219, DH104720, S2X25921, and our antibodies 1-20, 2-15, 2-7, 4-18, 5-7, and 10-4022-24. The footprints of mAbs with structures available were drawn in relation to the mutations found in B.1.1.529 RBD (Fig. 2a) and NTD (Fig. 2b). The risk to each of the 4 classes25 of RBD mAbs, as well as to the NTD mAbs, was immediately apparent. Indeed, neutralization studies on B.1.1.529 pseudovirus showed that 18 of the 19 mAbs tested lost neutralizing activity completely or partially (Fig. 2c and Extended Data Fig. 3). The potency of class 1 and class 2 RBD mAbs all dropped by >100-fold, as did the more potent mAbs in RBD class 3 (REGN10987, COV2-2130, and 2-7). The activity of S309 declined modestly, whereas Brii-198 was spared. All mAbs in RBD class 4 lost neutralization potency against B.1.1.529 by at least 10-fold, as did mAb directed to the antigenic supersite26 (4-18) or the alternate site23 (5-7) on NTD. Strikingly, all four combination mAb drugs in clinical use lost substantial activity against B.1.1.529, likely abolishing or impairing their efficacy in patients.
a, Footprints of RBD-directed antibodies, with mutations within B.1.1.529 highlighted in cyan. Approved or authorized antibodies are bolded. The receptor binding motif (RBM) residues are highlighted in yellow. b, Footprints of NTD-directed antibodies, with mutations within B.1.1.529 highlighted in cyan. The NTD supersite residues are highlighted in light pink. c, Neutralization of D614G and B.1.1.529 pseudoviruses by RBD-directed and NTD-directed mAbs. d, Neutralization D614G and B.1.1.529+R346K pseudoviruses by RBD-directed and NTD-directed mAbs.
Approximately 10% of the B.1.1.529 viruses in GISAID1 also contain an additional RBD mutation, R346K, which is the defining mutation for the Mu (B.1.621) variant27. We therefore constructed another pseudovirus (B.1.1.529+R346K) containing this mutation for additional testing using the same panel of mAbs (Fig. 2d). The overall findings resembled those already shown in Fig. 2c, with the exception that the neutralizing activities of S309 and Brii198 were further diminished or abolished. In fact, the entire panel of antibodies was essentially rendered inactive against this minor form of the Omicron variant.
The fold changes in IC50 of the mAbs against B.1.1.529 and B.1.1.529+R346K relative to D614G are summarized in the first two rows of Fig. 3a. The remarkable loss of activity observed for all classes of mAbs against B.1.1.529 suggest that perhaps the same is occurring in the serum of convalescent patients and vaccinated individuals.
a, Neutralization of pseudoviruses harboring single mutations found within B.1.1.529 by a panel of 19 monoclonal antibodies. Fold change relative to neutralization of D614G is denoted, with resistance colored red and sensitization colored green. b, Modeling of critical mutations in B.1.1.529 that affect antibody neutralization.
Key mutations conferring antibody resistance
To understand the specific B.1.1.529 mutations that confer antibody resistance, we next tested individually the same panel of 19 mAbs against pseudoviruses for each of the 34 mutations (excluding D614G) found in B.1.1.529 or B.1.1.529+R346K. Our findings not only confirmed the role of known mutations at spike residues 142-145, 417, 484, and 501 in conferring resistance to NTD or RBD (class 1 or class 2) antibodies4 but also revealed several mutations that were previously not known to have functional importance (Fig. 3a and Extended Data Fig. 4). Q493R mediated resistance to CB6 (class 1) as well as to LY-CoV555 and 2-15 (class 2), findings that could be explained by the abolishment of hydrogen bonds due to the long side chain of arginine and induced steric clashes with CDRH3 in these antibodies (Fig. 3b, left panels). Both N440K and G446S mediated resistance to REGN10987 and 2-7 (class 3), observations that could also be explained by steric hindrance (Fig. 3b, middle panels). The most striking and perhaps unexpected finding was that S371L broadly affected neutralization by mAbs in all 4 RBD classes (Fig. 3a and Extended Data Fig. 4). While the precise mechanism of this resistance is unknown, in silico modeling suggested two possibilities (Fig. 3b, right panels). First, in the RBD-down state, mutating Ser to Leu results in an interference with the N343 glycan, thereby possibly altering its conformation and affecting class 3 antibodies that typically bind this region. Second, in the RBD-up state, S371L may alter the local conformation of the loop consisting of S371-S373-S375, thereby affecting the binding of class 4 antibodies that generally target a portion of this loop (REF 10-40). It is not clear how class 1 and class 2 RBD mAbs are affected by this mutation.
Evolution of antibody resistance of SARS-CoV-2 variants
To gain insight into the antibody resistance of B.1.1.529 relative to previous SARS-CoV-2 variants, we evaluated the neutralizing activity of the same panel of neutralizing mAbs against pseudoviruses for B.1.1.78, B.1.52628, B.1.42929, B.1.617.29, P.130, and B.1.35131. It is evident from these results (Fig. 4 and Extended Data Fig. 5) that previous variants developed resistance only to NTD antibodies and class 1 and class 2 RBD antibodies. Here B.1.1.529, with or without R346K, has made a big mutational leap by becoming not only nearly completely resistant to class 1 and class 2 RBD antibodies, but also substantial resistance to both class 3 and class 4 RBD antibodies. B.1.1.529 is now the most complete “escapee” from neutralization by currently available antibodies.
Neutralization of SARS-CoV-2 variant pseudoviruses by a panel of 19 monoclonal antibodies. Fold change relative to neutralization of D614G is denoted.
Discussion
The Omicron variant struck fear almost as soon as it was detected to be spreading in South Africa. That this new variant would transmit more readily has come true in the ensuing weeks2. The extensive mutations found in its spike protein raised concerns that the efficacy of current COVID-19 vaccines and antibody therapies might be compromised. Indeed, in this study, sera from convalescent patients (Fig. 1c) and vaccinees (Figs. 1d and 1e) showed markedly reduced neutralizing activity against B.1.1.529. These findings are in line with emerging clinical data on the Omicron variant demonstrating higher rates of reinfection11 and vaccine breakthroughs. In fact, recent reports showed that the efficacy of two doses of BNT162b2 vaccine has dropped from over 90% against the original SARS-CoV-2 strain to approximately 40% and 33% against B.1.1.529 in the United Kingdom32 and South Africa33, respectively. Even a third booster shot may not adequately protect against Omicron infection32,34, but of course it is advisable to do so. Vaccines that elicited lower neutralizing titers35,36 are expected to fare worse against B.1.1.529.
The nature of the loss in serum neutralizing activity against B.1.1.529 could be discerned from our findings on a panel of mAbs directed to the viral spike. The neutralizing activities of all four major classes of RBD mAbs and two distinct classes of NTD mAbs are either abolished or impaired (Figs. 2c and 2d). In addition to previously identified mutations that confer antibody resistance4, we have uncovered four new spike mutations with functional consequences. Q493R confers resistance to some class 1 and class 2 RBD mAbs; N440K and G446S confer resistance to some class 3 RBD mAbs; and S371L confers global resistance to many RBD mAbs via mechanisms that are not yet apparent. While performing these mAb studies, we also observed that all the currently authorized or approved mAb drugs are rendered weak or inactive by B.1.1.529 (Figs. 2c and 3a). In fact, the Omicron variant that contains R346K seemingly flattens the antibody therapy landscape for COVID-19 (Fig. 2d and 3a).
The scientific community has chased after SARS-CoV-2 variants for a year. As more and more of them appeared, our interventions directed to the spike became increasingly ineffective. The Omicron variant has now put an exclamation mark on this point. It is not too far-fetched to think that this SARS-CoV-2 is now only a mutation or two away from being pan-resistant to current antibodies, either monoclonal or polyclonal. We must devise strategies that anticipate the evolutional direction of the virus and develop agents that target better conserved viral elements.
Methods
Serum samples
Convalescent plasma samples were obtained from patients with documented SARS-CoV-2 infection approximately one month after recovery or later. These samples were collected at the beginning of the pandemic in early 2020 at Columbia University Irving Medical Center, and therefore are assumed to be infection by the wild-type strain of SARS-CoV-24. Sera from individuals who received two or three doses of mRNA-1273 or BNT162b2 vaccine were collected at Columbia University Irving Medical Center at least two weeks after the final dose. Sera from individuals who received one dose of Ad26.COV2.S or two doses of ChAdOx1 nCov-19 were obtained from BEI Resources. Some individuals were also infected by SARS-CoV-2 in addition to the vaccinations they received. Note that, whenever possible, we specifically chose samples with high titers against the wild-type strain of SARS-CoV-2 such that the loss in activity against B.1.1.529 could be better quantified, and therefore the titers observed here should be considered in that context. All collections were conducted under protocols reviewed and approved by the Institutional Review Board of Columbia University. Additional information for the vaccinee samples can be found in Extended Data Table 1.
Monoclonal antibodies
Antibodies were expressed as previously described22, by synthesis of VH and VL genes (GenScript), transfection of Expi293 cells (Thermo Fisher), and affinity purification from the supernatant by rProtein A Sepharose (GE). REGN10987, REGN10933, COV2-2196, and COV2-2130 were provided by Regeneron Pharmaceuticals, Brii-196 and Brii-198 were provided by Brii Biosciences, CB6 was provided by Baoshan Zhang and Peter Kwong (NIH), and 910-30 was provided by Brandon DeKosky (MIT).
Variant SARS-CoV-2 spike plasmid construction
An in-house high-throughput template-guide gene synthesis approach was used to generate spike genes with single or full mutations of B.1.1.529. Briefly, 5’-phosphorylated oligos with designed mutations were annealed to the reverse strand of the wild-type spike gene construct and extended by DNA polymerase. Extension products (forward-stranded fragments) were then ligated together by Taq DNA ligase and subsequently amplified by PCR to generate variants of interest. To verify the sequences of variants, NGS libraries were prepared following a low-volume Nextera sequencing protocol37 and sequenced on the Illumina Miseq platform (single-end mode with 50 bp R1). Raw reads were processed by Cutadapt v2.138 with default setting to remove adapters and then aligned to reference variants sequences using Bowtie2 v2.3.439 with default setting. Resulting reads alignments were then visualized in Integrative Genomics Viewer40 and subjected to manual inspection to verify the fidelity of variants. Sequences of the oligos used in variants generation are provided in Extended Data Table 2.
Pseudovirus production
Pseudoviruses were produced in the vesicular stomatitis virus (VSV) background, in which the native glycoprotein was replaced by that of SARS-CoV-2 and its variants, as previously described24. Briefly, HEK293T cells were transfected with a spike expression construct with PEI (1 mg/mL) and cultured overnight at 37 °C under 5% CO2, and then infected with VSV-G pseudotyped ΔG-luciferase (G*ΔG-luciferase, Kerafast) one day post-transfection. Following 2 h of infection, cells were washed three times, changed to fresh medium, and then cultured for approximately another 24 h before supernatants were collected, centrifuged, and aliquoted to use in assays.
Pseudovirus neutralization assay
All viruses were first titrated to normalize the viral input between assays. Heat-inactivated sera or antibodies were first serially diluted in 96 well-plates in triplicate. Viruses were then added and the virus-sample mixture was incubated at 37 °C for 1 h. Vero-E6 cells (ATCC) were then added at a density of 3 × 104 cells per well and plates were incubated at 37 °C for approximately 10 h. Luciferase activity was quantified by using the Luciferase Assay System (Promega) according to the manufacturer’s instructions. Neutralization curves and IC50 values were derived by fitting a non-linear five-parameter dose-response curve to the data in GraphPad Prism version 9.2.
Authentic virus isolation and propagation
Authentic B.1.1.529 was isolated from a specimen from the respiratory tract of a COVID-19 patient in Hong Kong by Kwok-Yung Yuen and colleagues at the Department of Microbiology, The University of Hong Kong. Isolation of wild-type SARS-CoV-2 was previously described41. Viruses were propagated in Vero-E6-TMPRSS2 cells and sequence confirmed by next-generation sequencing prior to use.
Authentic virus neutralization assay
To measure neutralization of authentic SARS-CoV-2 viruses, Vero-E6-TMPRSS2 cells were first seeded in 96 well-plates in cell culture media (DMEM + 10% FBS + 1% penicillin/streptomycin) overnight at 37 °C under 5% CO2 to establish a monolayer. The following day, sera or antibodies were serially diluted in 96 well-plates in triplicate in DMEM + 2% FBS and then incubated with 0.01 MOI of wild-type SARS-CoV-2 or B.1.1.529 at 37 °C for 1 h. Afterwards, the mixture was overlaid onto cells and further incubated at 37 °C under 5% CO2 for approximately 72 h. Cytopathic effects were then visually assessed in all wells and scored as either negative or positive for infection by comparison to control uninfected or infected wells in a blinded manner. Neutralization curves and IC50 values were derived by fitting a non-linear five-parameter dose-response curve to the data in GraphPad Prism version 9.2.
Antibody footprint analysis and RBD mutagenesis analysis
The SARS-CoV-2 spike structure used for displaying epitope footprints and mutations within emerging strains was downloaded from PDB (PDBID: 6ZGE). The structures of antibody-spike complexes were also obtained from PDB (7L5B for 2-15, 6XDG for REGN10933 and REGN10987, 7L2E for 4-18, 7RW2 for 5-7, 7C01 for CB6, 7KMG for LY-COV555, 7CDI for Brii-196, 7KS9 for 910-30, 7LD1 for DH1047, 7RAL for S2X259, 7LSS for 2-7, and 6WPT for S309). Interface residues were identified using PISA42 using default parameters. The footprint for each antibody was defined by the boundaries of all epitope residues. The border for each footprint was then optimized by ImageMagick 7.0.10-31 (https://imagemagick.org). PyMOL 2.3.2 was used to perform mutagenesis and to make structural plots (Schrödinger).
Author contributions
D.D.H. conceived this project. L.H.L., S.I., and M.W. conducted pseudovirus neutralization experiments. J.F-W.C., H.C., K.K-H.C., T.T-T.Y., C.Y., K.K-W.T., and H.C. conducted authentic virus neutralization experiments. Y.G. and Z.Z. conducted bioinformatic analyses. L.Y.L. and Y.M.H. constructed the spike expression plasmids. Y.L. managed the project. J.Y. expressed and purified antibodies. M.T.Y. and M.E.S. provided clinical samples. M.S.N. and Y.X.H. contributed to discussions. H.H.W., K-Y.Y., and D.D.H. directed and supervised the project. L.H.L., S.I., and D.D.H. analyzed the results and wrote the manuscript.
Competing interests
L.L., S.I., M.S.N., J.Y., Y.H., and D.D.H. are inventors on patent applications on some of the antibodies described in this manuscript.
Data and materials availability
All data are provided in the paper or the Supplementary Information.
Acknowledgements
We are grateful Regeneron Pharmaceuticals, B. Zhang and P. Kwong (NIAID), and B. Dekosky (MIT) for antibodies. This study was supported by funding from the Gates Foundation, JPB Foundation, Andrew and Peggy Cherng, Samuel Yin, Carol Ludwig, David and Roger Wu, Health@InnoHK, and the National Science Foundation (MCB-2032259).