key: cord-0840844-5hr11ccv
authors: Dejnirattisai, Wanwisa; Huo, Jiandong; Zhou, Daming; Zahradník, Jiří; Supasa, Piyada; Liu, Chang; Duyvesteyn, Helen M.E.; Ginn, Helen M.; Mentzer, Alexander J.; Tuekprakhon, Aekkachai; Nutalai, Rungtiwa; Wang, Beibei; Dijokaite, Aiste; Khan, Suman; Avinoam, Ori; Bahar, Mohammad; Skelly, Donal; Adele, Sandra; Johnson, Sile Ann; Amini, Ali; Ritter, Thomas; Mason, Chris; Dold, Christina; Pan, Daniel; Assadi, Sara; Bellass, Adam; Omo-Dare, Nikki; Koeckerling, David; Flaxman, Amy; Jenkin, Daniel; Aley, Parvinder K.; Voysey, Merryn; Costa Clemens, Sue Ann; Naveca, Felipe Gomes; Nascimento, Valdinete; Nascimento, Fernanda; Fernandes da Costa, Cristiano; Resende, Paola Cristina; Pauvolid-Correa, Alex; Siqueira, Marilda M.; Baillie, Vicky; Serafin, Natali; Kwatra, Gaurav; Da Silva, Kelly; Madhi, Shabir A.; Nunes, Marta C.; Malik, Tariq; Openshaw, Peter JM.; Baillie, J Kenneth; Semple, Malcolm G.; Townsend, Alain R.; Huang, Kuan-Ying A.; Tan, Tiong Kit; Carroll, Miles W.; Klenerman, Paul; Barnes, Eleanor; Dunachie, Susanna J.; Constantinides, Bede; Webster, Hermione; Crook, Derrick; Pollard, Andrew J.; Lambe, Teresa; Paterson, Neil G.; Williams, Mark A.; Hall, David R.; Fry, Elizabeth E.; Mongkolsapaya, Juthathip; Ren, Jingshan; Schreiber, Gideon; Stuart, David I.; Screaton, Gavin R.
title: SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses
date: 2022-01-04
journal: Cell
DOI: 10.1016/j.cell.2021.12.046
sha: a2b5d6d98b20727b322a16db7c66c408652c451e
doc_id: 840844
cord_uid: 5hr11ccv

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate Omicron-B.1.1.529 was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses.

Since the end of 2020 a series of viral variants have emerged in different regions where some have caused large outbreaks, Alpha and more recently Delta have had the greatest global reach, whilst Beta , Gamma and Lambda (Colmenares-Mejia et al., 2021) , although causing large outbreaks in Southern Africa and South America, did not become dominant in other parts of the World. Indeed, Beta was later displaced by Delta in South Africa.

The rapid emergence of Omicron (https:www.who.int/news/item/26-11-2021-classificationof-omicron-(bi1.1.529)-sars-cov-2-variant-of-concern) on the background of high Beta immunity implies that the virus may have evolved to escape neutralization by Beta specific J o u r n a l P r e -p r o o f serum . Within S, Omicron has 30 substitutions plus the deletion of 6 and insertion of 3 residues, whilst in all the other proteins there are a total of 16 substitutions and 7 residue deletions. Particular hotspots for the mutations are the ACE2 receptor binding domain (RBD) (15 amino acid substitutions) and the N-terminal domain (NTD) (3 deletions totalling 6 residues, 1 insertion, 4 substitutions).

S mediates cellular interactions. It is a dynamic, trimeric structure Walls et al., 2017; Wrapp et al., 2020) which can be lipid bound (Toelzer et al., 2020) and tightly associated in a 'closed' form or unfurled to expose one or more RBDs allowing both receptor binding and increased access to neutralising antibodies. Once bound to a cell, S undergoes cleavage and a drastic elongation converting it to the post fusion form.

Most potent neutralizing antibodies target the ACE2 footprint Lan et al., 2020; Liu et al., 2021b) occupying ~880 A 2 at the outermost tip of the RBD (the neck and shoulders referring to the torso analogy ), preventing cell attachment. A proportion of antibodies are able to cross-neutralize different variants ) and a few of these bind to a motif surrounding the N-linked glycan at residue 343 Liu et al., 2021b) . These antibodies, exemplified by S309 (Pinto et al., 2020) can cross-react with SARS-CoV-1 but do not block ACE2 interaction and their mechanism of action may be to destabilize the S-trimer. Neutralizing anti-NTD mAbs do not block ACE2 interaction and bind to a so-called supersite on the NTD Chi et al., 2020) , however they generally fail to give broad protection as the supersite is disrupted by a variety of NTD mutations present in the variants of concern. Moreover, some NTD J o u r n a l P r e -p r o o f binding antibodies were shown to have an infectivity-enhancing effect by induction of S open state (Liu et al., 2021c) .

In this report, we study the neutralization of Omicron by a large panel of sera collected from convalescent early pandemic, Alpha, Beta, Gamma and Delta infected individuals, together with vaccinees receiving three doses of the Oxford/AstraZeneca (AZD1222) or the Pfizer BioNtech (BNT16b2) vaccines. There is widespread reduction in the neutralization activity of serum from multiple sources and we use these data to plot an antigenic map where Omicron is seen to occupy the most distant position from early pandemic viruses which form the basis for current vaccines.

We show that Omicron escapes neutralization by the majority of potent monoclonal antibodies (mAbs) arising after both early pandemic and Beta variant. Utilizing a large bank of structures (n=29) from panels of potent monoclonal antibodies we describe the mechanism of escape caused by the numerous mutations present in Omicron RBD, which includes most mAbs developed for prophylactic or therapeutic use (Baum et al., 2020) .

Analysis of the binding of ACE2 to RBD and structural analysis of the Omicron RBD indicates that changes at residues 498 and 501 of the RBD have locked ACE2 binding to the RBD in that region sufficiently strongly to enable the generation of a plethora of less favourable changes elsewhere, providing extensive immune escape and in the process resulting in a final net affinity for ACE2 similar to the early pandemic virus.

Omicron has changes throughout its proteome but S changes dominate, with 30 amino acid substitutions plus 6 residues deleted and 3 inserted (Figure 1 and 2) . Ten of these were found previously in at least two lineages (D614G was mutated early on and maintained throughout).

Of those ten, six have the same amino-acid substitution in >75% of the sequences, and only one (E484A) has a unique substitution in Omicron (in Beta and Gamma it is a Lys). Figure S1A shows the number of mutant sequences per residue at positions undergoing mutations in independent lineages. This can be interpreted in two ways, one is that the later mutations are epistatic to one another and thus are more difficult to reach, or that they do not contribute to virus fitness.

The Omicron RBD has 15 changes in total in the RBD, described below. The NTD also has numerous changes, including 4 amino acid substitutions, 6 amino acids deleted and 3 amino acids inserted, also described below. Several mutations found in Omicron occur in residues conserved in SARS-CoV-1 and many other Sarbecoviruses (Figure 1) . These observations agree with the Pango classification (Rambaut et al., 2020) which places Omicron at a substantial distance from all other variants.

The Alpha variant has a single change in the RBD at N501Y (Figure 2D ) which occupies the right shoulder and contributes to the ACE2 binding footprint. Beta has two further mutations in the RBD: K417N and E484K, at back of the neck and left shoulder respectively ( Figure 2E ), also part of the ACE2 footprint ( Figure 2C ) .

Gamma mutations are similar: K417T, E484K, N501Y . Delta mutations, L452R front of neck, T478K far side of left shoulder, fall just peripheral to the ACE2 binding footprint ( Figure 2F ) . All of these variants have at least one RBD mutation in common with Omicron. Of the 15 Omicron changes in the RBD, nine map to the ACE2 binding footprint: K417N, G446S, S477N, E484A, Q493R, G496S, Q498R, N501Y, Y505H with N440K and T478K just peripheral (Figure 2B-C) . Aside from these, mutations occur on the right flank: G339D, S371L, S373P and S375F (Figure 2B) , the last three of which are adjacent to a lipid binding pocket ( Figure S1B ) (Toelzer et al., 2020) . The lipid binding pocket has been seen occupied by a lipid similar to linoleic acid in an unusually rigid state of S where all RBDs are found in a locked-down configuration stabilised by lipid-bridged quaternary interactions between adjacent RBDs. However, this lipid bound form has been rarely seen, instead the pocket is usually empty and collapsed, with the RBD alternating between looser down and up conformations. We presume that this is because the pocket readily empties of lipid during protein purification, indeed rapidly prepared virus particles tend to have the RBDs closer to the locked down state . Loss of lipid promotes RBD presentation to the target cell.

Until now the antigenic properties of variant viruses have been well described by assuming each change produces only a local change in structure and use this assumption to rationalising the serological impact of the changes in Omicron. We present structural data later to qualify this assumption.

The mutations seen in the NTD lie on exposed flexible loops, which differ from those in SARS-CoV-1 and are likely antigenic ( Figure 1A) . The pattern of deletions and insertions seen in Omicron consistently changes those loops that are most different from SARS-CoV-1 to being more SARS-CoV-1-like, at least in length. Of the N1, N3 and N5 loops which comprise the antibody supersite, Omicron has a substitution at G142D and deletion of residues 143-145 in N3 which would mitigate against binding by a number of potent neutralizing antibodies e.g.

4A8 and 159 (Chi et al., 2020; Dejnirattisai et al., 2021b) . The deletion of residues 69 and 70 in N2 has also occurred in the Alpha variant whilst the deletion at residue 211, substitution at 212 and insertion at 214 are unique to Omicron. All these changes are on the outer surface and likely antigenic.

We isolated Omicron virus from the throat swab of an infected case in the UK. Following culture in VeroE6 cells transfected with TMPRSS2 the S gene sequence was confirmed to be the Omicron consensus with the additional mutation A701V, which is present in a small number of Omicron sequences.

We have collected convalescent serum/plasma, with the indicated median day of sampling, from individuals infected early in the pandemic (n=32, median day 42) before the emergence of the variants of concern (VOC), along with cases infected with Alpha (n=18, median day 18), Beta (n=14, median day 61), Gamma (n=16, median day 63) and Delta (n=42, median day 38).

Neutralization assays were performed against Omicron and compared with neutralization titres for Victoria (an early pandemic strain), Alpha, Beta, Gamma and Delta.

In all cases neutralization titres to Omicron were substantially reduced compared to either the ancestral strain Victoria or to the homologous strain causing infection and in a number of cases immune serum failed to neutralize Omicron at 1/20 dilution (Figure 3A-E In summary, Omicron causes widespread escape from neutralization by serum obtained following infection by a range of SARS-CoV-2 variants meaning that previously infected individuals will have little protection from infection with Omicron, although it is hoped that they will still maintain protection from severe disease.

We have collected sera from Delta infected cases and because Delta spread in the UK during the vaccination campaign, we obtained sera from three different groups; Delta infection only (n=19) (Figure 3E ), Delta infection following vaccination (n=9) or vaccination following Delta infection (n=8) ( Figure 3F ). Neutralization assays against early pandemic, Alpha, Beta, Gamma, Delta and Omicron viruses were performed. Compared to Delta-only infected individuals, sera from cases who had received vaccine and been infected by Delta showed substantially higher neutralization to all viruses tested; early pandemic, with Delta infected and vaccinated sera showing a 7.9-fold (p<0.0001) increase in the neutralization of Omicron, J o u r n a l P r e -p r o o f compared to Delta infection alone. To confirm the boosting effect of vaccination we collected a paired blood sample from 6 Delta cases before and after vaccination which clearly demonstrates the boosting effect of infection and vaccination ( Figure S2 ).

In a number of countries booster programmes have been launched to counter waning immunity and the increasing frequency of break-through infections with Delta. To examine the effect of booster vaccination, we tested neutralization of Victoria, Delta and Omicron viruses using sera from individuals receiving 3 doses of ADZ1222 (n=41) or BNT162b2 (n=20).

For ADZ1222, serum was obtained 28 days following the second and third doses ( Figure 3G ).

For BNT162b2, serum was obtained 28 days, 6 months, immediately prior to the third dose and 28 days following the third dose ( Figure 3H ).

At 28 days following the third dose, for ADZ1222, the neutralization titre to Omicron was reduced 12.7-fold (p<0.0001) compared to Victoria and 3.6-fold (p<0.0001) compared with Delta; for BNT162b2, the neutralization titre to Omicron was reduced 14.2-fold (p<0.0001) compared to Victoria and 3.6-fold (p<0.0001) compared to Delta. The neutralization titres for Omicron were boosted 2.7-fold (p<0.0001) and 34.2-fold (P<0.0001) following the third dose of ADZ1222 and BNT162b2 respectively compared to 28 days following the second dose. Of concern, and as has been noted previously, neutralization titres fell substantially between 28 days and 6 months following the second dose of the BNT162b2 vaccine, although we did not measure titres 6 months following the second dose of AZD1222.

J o u r n a l P r e -p r o o f

In summary, neutralization titres against Omicron are boosted following a third vaccine dose, meaning that the campaign to deploy booster vaccines should add considerable protection against Omicron infection.

We have previously reported a panel of 20 potent neutralizing antibodies (FRNT50 < 100ng/ml) isolated from cases infected with early pandemic viruses (Wuhan) . Neutralization assays against Omicron were performed and compared with neutralization of early pandemic, Alpha, Beta, Gamma, Delta viruses; 17/20 mAbs failed to neutralize Omicron (FRNT50 >10g/ml) whilst the titres for mAbs 58, 222 and 253 were reduced 3.4, 12.6 and 19.3 -fold compared to Victoria (Figure 4 , Table S1 ).

The binding sites of these antibodies were mapped together with other published structures to 5 epitopes (based on the position of the centre of gravity of each antibody) either by direct structural studies or competition analyses . According to the torso analogy these were designated: neck left shoulder, right shoulder, right flank and left flank ( Figure 2B ). In Figure 5A -D we show the mapping of the density of centroids to the surface of the RBD with the Omicron mutations shown as spikes (the information is also mapped to the primary structure in Figure 1A ) and selected antibody binding is shown schematically in Figure 5E -G. As expected there is correlation between the two, although the antibody centroids are more broadly spread across the RBD surface, in particular there are no mutations in the left flank epitope, where a significant number of antibodies bind ( Figure 5A ). These antibodies can neutralize in some assays, and confer protection (Barnes et al., 2020; Dejnirattisai et al., 2021a , Huang et al.. 2021 ) and this cryptic J o u r n a l P r e -p r o o f epitope might therefore be an important target for therapeutic antibody applications and cross-protective vaccine antigen (Pinto et al., 2020) . We demonstrate the continued binding of this class of antibodies below.

Nineteen of the 20 most potent (FRNT50<100ng/ml) neutralising monoclonal antibodies mapped to the ACE2 binding site across the neck and shoulder epitopes of the RBD and 5 of these classified as public IGVH3-53 antibodies Yuan et al., 2020) .

Mapping these onto the RBD surface ( Figure 5B ) shows that the centroids are highly concentrated in the neck region. IGVH3-53 mAbs were especially common in early pandemic responses and although their centroid is at the neck they are orientated such that their light chain CDRs interact with the right shoulder ( Figure S3 ). Most IGVH3-53 mAbs are sensitive to the N501Y mutation, although some such as mAb 222 or Beta-27 can still neutralize 501Y

containing viruses Liu et al., 2021b) . Omicron mutation Y505H makes a direct interaction with the L1 and L3 CDRs of mAb 222 and together with Q493R is likely responsible for the 12.6-fold reduction in the neutralization titre of mAb 222 ( Figure 4A , Table S1 ).

MAbs 253, 55 and 165 are IGVH1-58 mAbs which bind an epitope towards the left shoulder.

H3 contacts S477N and Q493R is likely disruptive ofH2 interactions, leading to the 19.3-fold reduction in neutralization( Figure S3 ).

The neutralizing activity of mAbs 88, 316, and 384 is knocked out for Omicron ( Figure 4A , Table S1 ), all interact with E484 (mAb 316 via H1 and H2) within the left shoulder epitope and the E484A mutation isis unfavourable. For mAb 316, Q493R will also likely be deleterious due J o u r n a l P r e -p r o o f to contacts with H1 and H3. Broadly neutralizing mAb 58 binds at the front of the RBD reaching towards the right flank in an area which is relatively clear of mutations and thus is unaffected ( Figure S3 ). MAb 278 binds more of the right shoulder, with L3 in contact with G446, and the G446S mutation in Omicron knocks out activity ( Figure S3) .

MAb 170 will be affected by Q493R and Q498R, which directly interact with L1 and H3 respectively ( Figure S3 ). Q498R is between G496S and G446S ( Figure 2B ) and G446 is in proximity to H1 and together these mutations knock out the activity of mAb 170 ( Figure 4A , Table S1 ). The binding sites of selected potent antibodies are shown in Figure 5E . All of these, with the exception of mAb58, are affected by the mutations in Omicron. To understand the resilience of nAb58 we determined the structure of a ternary complex of early pandemic RBD with Fabs for mAbs 58 and 158 (Table S2) confirming that its epitope includes no residues mutated in Omicron ( Figure S3 ).

We have derived a panel of 27 potent Beta antibodies (FRNT <100 ng/ml) and this revealed a surprisingly skewed response with 18/27 potent antibodies targeting the Beta mutations: E484K, K417N and N501Y. This is seen in Figure 5C , where the focus on residues in the shoulders has spread the centroid patch out towards several Omicron mutation sites, this information is mapped to the primary structure in Figure 1A and a schematic of the binding of the four potent cross-reactive antibodies in Figure 5F . Whilst K417N and N501Y are conserved in Omicron, E484 is mutated to an alanine, which seems a likely escape mutation from either 484E (early pandemic/Alpha) or 484K (Beta).

Neutralization assays were performed against Omicron and show a complete loss of activity for 17/27 Beta mAb ( Figure 4B , Table S1 ). Substantial reductions in neutralization titres were observed for many of the rest of the Beta panel, with 29, 40, 47, 53, 54, 55 and 56 able to neutralize Omicron with titres < 400ng/ml.

A large number of Beta mAbs target the 501Y mutation including a public antibody response mediated through IGVH4-39 (n=6) and the related IGVH4-30 (n=1) and many are likely to be sensitive to the numerous mutations in this region: N440K, G446S, Q493R, G496S, Q498R and Y505H. In total 11 antibodies make contact with 501Y; 10, 23, 24, 30, 40, 54, 55, 56, and 29 bind epitopes dependent on 417N/T together with 501Y, (antibodies in italic are VH4-39 or 4-30 and the neutralization of Omicron for those in bold is completely knocked out). Beta MAbs targeting the back of the neck epitope (Beta-22,29,30) will be affected, for example in the case of Beta-29, H1 makes extensive interactions with residues Q493, G496 and Y505 ( Figure S4 ). Beta-44 binding to the left shoulder epitope has already been shown to be sensitive to T478K whilst the combination of S477N and T478K

in Omicron is likely to be more deleterious. Interestingly, several of the antibodies (Beta 40, 54, 55, 56 and 22, 29 (501Y 417N/T)) retain some activity and this is explained below with reference also to the structure of the Omicron RBD/Fab 55 complex.

Four Beta-mAbs potently cross-neutralize all Alpha, Beta, Gamma and Delta variants , Figure 5F ). Of these, Beta-27 is a VH3-53 antibody, which contacts Q493 and Y505

in a similar way to mAb222 and shows reduced neutralization of Omicron ( Figure 4B , Table   S1 ). Beta-47, a VH1-58 antibody, has contacts with S477 and Q493 likely leading to the observed reduction in neutralization of Omicron.

Beta-49 and 50 belong to the IGVH1-69 gene family, bind similarly to the right flank and are knocked out by Omicron ( Figure 4B , Table S1 ). They lie directly above RBD G399 and would clash with G399D. Beta-53 also binds to the right flank with H1 contacting residue 339 and likely clashing with G339D and L1 likely contacts G446S leading to the observed two log reduction in Omicron neutralization compared to Beta ( Figure S4 ).

Various individual antibodies or cocktails of antibodies (usually recognizing different epitopes to reduce risk of escape (Sun et al., 2021) ) have been licensed for use and the aggregate of their binding shown in Figure 5G . This illustrates the strong correlation of binding with sites of mutation (this is mapped to the primary structure in Figure 1A ) and neutralisation of Omicron is markedly reduced in most( Figure 4C , Table S1 ). Specifically: LY-CoV016 and 555: Activity of both antibodies on Omicron is knocked out. LY-CoV016 is a VH3-53 antibody and extensive interactions with N501 and Y505 via L1 and L3 make it vulnerable to mutations at these residues ( Figure 5G , S5). LY-CoV555 (Sun and Ho, 2020) is sensitive to the E484K mutation in delta and also contacts Q493.

AllAdagio antibodies suffer considerable loss of activity against Omicron ( Figure 4C ). Activity of ADG10 and ADG30 being completely lost whilst ADG20 activity is reduced 276-fold.

Fitness of a virus can stem from higher infectivity or evasion of the immune system. One way to identify mutations that increase binding affinity is by selection using a randomly mutated RBD displayed on the yeast surface for ACE2 binding, to obtain the highest affinity clone RBD-62. Mutations fixed for higher affinity binding included N501Y, E484K, S477N and most J o u r n a l P r e -p r o o f prominently, Q498R ( Figure 6A ) (Zahradnik et al., 2021b) . Interestingly, Q498R was selected only at later stages. This is explained by the 2-fold reduction in affinity as a single mutation ( Figure 6A ). However, in combination with the N501Y mutation, the affinity is increased 26fold, more than any other mutation analyzed. Adding to this the S477N mutation, one obtains a 37-fold increase in binding ( Figure 6B ). These three mutations, selected through in vitro evolution, were found together for the first time in the Omicron variant.

We measured the affinity of Omicron RBD for ACE2 using SPR and yeast display titration.

Perhaps surprisingly, the affinity was on a par with that of the early virus, 8 nM and 7 nM respectively using SPR ( Figure 6B and S6A) and 2.9 nM and 1.9 nM using yeast display titration (SPR and yeast display titration data strongly correlate, but with a constant shift in absolute values (Zahradnik et al., 2021b) ). This implies that the increased affinity imparted by S477N, Q498R and N501Y are being offset by other mutations in the ACE2 footprint. We measured the affinities of the other single mutations in the ACE2 binding footprint of Omicron (using yeast display titration), shown in Figure 6B , C, and they provide a rationale for this.

T478K in the presence of N501Y decreased the positive effect of the latter by 2-fold. Y505H

reduces binding of Q498R, N501Y double mutant by 50%. G496S and the triple-mutation S371L, S373P and S375F reduce binding by 2-fold and 2.2-fold respectively. The effect of changing the triple-mutant (S371L, S373P and S375F) back to the wild-type sequence was even more pronounced on the background of Omicron, where the affinity increased from 2.9 nM to 0.4 nM (7-fold). Moreover, this back-to-wild-type triple-mutant increased the expression on the surface of yeast 10-fold relative to Omicron. This indicates a functional role in increasing the fitness of the virus for this triple-mutant, which requires the binding enhancement provided by the Q498R, N501Y double mutant. E484A (instead of the Lys found J o u r n a l P r e -p r o o f other variants, Figure 6A ) was neutral. While K417N (found in the Beta variant) on its own decreases binding substantially, the effect on binding when combined with other mutations is smaller ( Figure 6B ). Two single-mutations found specifically in Omicron, Q493R and N440K did increase binding, probably due to increasing the electrostatic complementarity between ACE2 (negatively charged) and the RBD (positively charged) ( Figure 6D ).

Comparing the structure of the complex of the pM affinity RBD-62 with ACE2 (Zahradnik et al., 2021b, PDB:7BH9) to that of Omicron RBD bound to Beta 55 antibody (described below, see Table S2 , Figure 6C ), shows high similarity, with an RMSD of 0.55 Å over 139 residues.

Importantly, the locations of the binding enhancing mutations 477N, 498R and 501Y are conserved between the two, despite the RBD-62 bound to ACE2, while Omicron RBD is not.

This shows that these residues are pre-arranged for tight binding, implying low entropic penalty of binding.

We used the matrix of neutralization data generated in Figure 

We firstly used Alphafold2 (Jumper et al., 2021) to predict the Omicron RBD structure ( Figure   S1B ). The top ranked structure was very similar to the early pandemic RBD (RMSD for C 0.71 Å, residues 334 to 528), with a a significant difference in the region of the triple serine mutations 371-375, on the right flank ( Figure S1B ). We then determined the high-resolution crystal structure of the Omicron RBD domain in complex with two Fabs: Beta55 and EY6A, Figure S6B , Table S2 (Huang et al., 2021 Zhou et al., 2020) . The RBD structure is indeed close to that of early pandemic viruses (RMSD 0.9 Å for 187 C) with the only significant change at the 371/373/375 triple serine mutations ( Figure 7E ). The rearrangement in this region is essentially an amplified version of that predicted by Alphafold2, suggesting that such algorithms have some value in predicting the effect of dense mutations as seen in Omicron RBD. The mutations S371L, S373P, S375F all change from small flexible polar serine residues to bulkier, less flexible hydrophobic residues. Interestingly, all the Omicron S mutations involve single codon changes apart from S371L which requires two changes from TCC to CTC indicative of underlying strong selection pressure and functional change. Although the rearrangement in Omicron is quite modest it is exactly this region of the structure that undergoes a larger conformation change when lipid is bound into the pocket ( Figure S1B) .

Changes in the serine rich loop allow the attached helix to swing out, opening the pocket for lipid binding. It is possible that increased rigidity and the entropic penalty of exposing J o u r n a l P r e -p r o o f hydrophobic residues may disfavour lipid binding to Omicron, which would alter the properties of the virus, explaining the selection of these changes.

The binding of EY6A to the left flank of the RBD is essentially unchanged from that observed previously ) (KD 7.8 and 6.8 nM for early pandemic and Omicron RBDs respectively by SPR) (Figure S6A, 7F) , in line with this cryptic epitope, which is highly conserved for functional reasons, being a good target for broadly neutralising therapeutic antibodies.

Beta55, as predicted earlier , binds to the right shoulder, around residue 501.

Interestingly, the epitope includes several residues mutated in Omicron from the early pandemic virus (including Q498R, N501Y, Y505H) ( Figure 7E, S6B, S6C) . It is remarkable that despite these significant changes, neutralization is relatively little affected. The neutralisation result was confirmed by measurements of the binding affinity, 177 nM and 204 pM for the early pandemic and Omicron RBDs respectively ( Figure S6A ). To confirm the structural basis.

we also determined the crystal structure of an analogous ternary complex formed with early pandemic RBD (Table S2) , as expected the details of the interaction are essentially identical.

If we extend the analysis of the 501Y targeting antibodies by comparing the structures of Beta-6, 24, 40 and 54, we find subtle explanations, thus Beta-24 and some others, are knocked out due to a clash with CDR-L1 created by the Q493R mutation ( Figure S6D) , whereas for antibodies Beta-40, 54 and 55 this mutation can be accommodated. In addition, the Q498R mutation may create a hydrogen bond in Beta-40 or a salt bridge in Beta-54 to CDR-H3, which may compensate for the loss of binding affinity due to changes around residue 501 ( Figure   S6E ). Thus, the surprising resilience of several of the 501Y targeting antibodies may be J o u r n a l P r e -p r o o f because the mutated residues in this region are not 'hot-spots' of interaction and mutations can sometimes be accommodated without significant impact on affinity. This may suggest that a major driver for evolution was the less 501-focussed responses to early viruses.

The first 4 Omicron sequences were deposited on 24 th November 2021. Within days distant international spread was seen and it is causing great concern due to its high transmissibility and ability to infect previously exposed or vaccinated individuals. Only three weeks after the virus was first detected in the UK Omicron cases outnumbered Delta in London and the number of daily new cases in the UK is larger than at any previous time in the pandemic. Over the next weeks disease severity will become clearer.

The density of mutational changes (including deletions and insertions) found in Omicron S is extraordinary, being more than three times that observed in previous variants. Within S, as observed for other variants, the NTD, RBD and the furin cleavage site region are hotspots for mutation (Zahradnik et al., 2021b) and within the RBD, mutations are concentrated on the ACE2 interacting surface and the right flank.

Most potent neutralizing antibodies bind on, or in close proximity to the ACE2 footprint (neck and shoulder epitopes) and block interaction of S with ACE2, thereby preventing viral attachment to the host cell. There are two other classes of potent neutralizing mAbs, firstly antibodies binding in close proximity to the N343 glycan (right flank epitope) exemplified by Vir S309 (Pinto et al., 2020) which include the Beta 49, 50 and 53 antibodies J o u r n a l P r e -p r o o f used in our analysis. These mAbs bind distant from the ACE2 binding site, do not block ACE2

interaction and their mechanism of action may be to destabilize the S trimer. Finally, antibodies binding to the supersite on the NTD can also be potently neutralizing although the mechanism of action of NTD antibodies remains obscure Chi et al., 2020; Dejnirattisai et al., 2021a) . Multiple mutations at all three of these sites: the receptor binding site, proximal to N343 glycan and NTD are found in Omicron, and lead to substantial reduction in neutralization titres for naturally immune or vaccine sera, with many showing complete failure of neutralization. This together with the widespread failure of potent mAb to neutralize Omicron point to a driver of immune evasion for their evolution.

The left flank epitope, which is not mutated in Omicron, is used by antibodies that do not block ACE2 binding, but are protective (Barnes et al., 2020; Dejnirattisai et al., 2021a; Hastie et al., 2021; Huang et al., 2021; Zhou et al., 2020) . Here we demonstrate structurally and by affinity measurements that this epitope is conserved unchanged in Omicron.

Following repeated rounds of selection by yeast display for high ACE2 affinity, RBD-62 (I358F, V445K, N460K, I468T, T470M, S477N, E484K, Q498R, N501Y) emerged as the highest affinity clone with a 1000-fold increase in affinity for ACE2 from 17 nM for Wuhan RBD to 16 pM for RBD-62. It is striking that the key contributors for the high affinity of RBD-62 are present in Omicron. Interestingly, the combination of mutations K417M, E484K, Q493R, Q498R and N501Y also emerged after 30 passages in mouse lungs (Wong et al., 2021) . This mouseadapted virus was highly virulent and caused a more severe disease. The appearance of E484K, Q493H/R, Q498R and N501Y in yeast display and mouse adaptation experiments is a strong indication that the tighter binding to ACE2 also facilitates more efficient transmission.

However, in Omicron overall affinity for ACE2 is not increased, suggesting a different strategy.

Since mutations S477N, Q498R and N501Y are likely to increase ACE2 affinity by 37-fold, we hypothesise that these changes, also found in RBD-62, serve to anchor the RBD to ACE2, leaving the rest of the receptor binding motif more freedom to develop further mutations, including those that reduce ACE2 affinity, in the quest to evade the neutralizing antibody response. Indeed, K417N, T478K, G496S, Y505H and the triple S371L, S373P, S375F reduce affinity to ACE2, while driving immune evasion. All this is achieved with very minimal structural changes in the isolated Omicron RBD ( Figure 7E ).

These observations provide a valuable lesson on the plasticity of protein-protein binding sites, maintaining nM binding affinity (Cohen-Khait and Schreiber, 2016). Thus, whilst the extreme concentration of potent neutralizing antibodies around the 25 amino acid receptor footprint of ACE2 suggest that this would be an Achilles heel for SARS-CoV-2, with ACE2 placing constraints on its variability (with receptor binding sites therefore sometimes hidden (Rossmann et al., 1985) ). In practice, the extraordinary plasticity of this site to absorb mutational change, whilst retaining affinity for ACE2 is a potent weapon to evade the antibody response. Such camouflage of receptor binding sites has been observed before (see for example (Acharya et al., 1989) , but it seems that by acquiring a lock on the ACE2 receptor at one point, through 498 and associated mutations many other less energetically favorable changes can be tolerated, fueling antigenic escape . Thus, by mutating the receptor binding site, the virus can modulate ACE2 affinity and potentially transmissibility, whist at the same time evading the antibody response.

How Omicron evolved is under debate. The results presented here show that immune evasion is a primary driver in its evolution, sacrificing the affinity enhancing mutations for optimizing immune evading mutations. This could for instance happen by a combination of a single immunocompromised individual which further evolved in rural, unmonitored populations (Clark et al., 2021) . Virus evolution has been previously observed in chronically infected HIV+ individuals and other immunocompromised cases leading to the expression of the N501Y, E484K and K417T mutations Karim et al., 2021; Kemp et al., 2021) . What seems beyond doubt from the ratio of nonsynonymous to synonymous mutations (only one synonymous mutation in all of S) is that the evolution has been driven by strong selective pressure on S. It has been predicted that increasing immunity by natural infection or vaccination will increase the selective pressure to find a susceptible host, either by increased transmissibility or antibody evasion, it appears that Omicron has achieved both of these goals although our data only speak directly to antibody evasion.

In addition to changes in the ACE2 footprint Omicron RBD possesses a triplet of mutations from serines to more bulky, hydrophobic residues, a motif not found in any other

Sarbecoviruses. This introduces structural changes and may led to loss of ability to form the lipid binding pocket which might normally aid release of the virus from infected cells. One of these mutations requires a double change in the codon reinforcing its significance, and it is conceivable there is synergy with the change at residue 498, perhaps explaining why this mutation has not established itself earlier.

For most mAbs the changes in interaction are so severe that activity is completely lost or severely impaired. This also extends to the set of mAbs developed for clinical use, the activity J o u r n a l P r e -p r o o f of most is lost, AZD8895 and ADG20 activity is substantially reduced while the activity of Vir S309 is more modestly reduced.

Omicron has now got a foothold in many countries, in the UK it has an estimated doubling time of 2.5 days, 2 doses of vaccine appear to give low protection from infection, while 3 doses give better protection. There is considerable concern that Omicron will rapidly replace Delta and cause a large and sharp peak of infection in early 2022. It is likely that substantial increased transmissibility and immune evasion are contributing to the explosive rise in Omicron infections. At present, the only option to control the spread of Omicron, barring social distancing and mask wearing, is to pursue vaccination with Wuhan containing antigen, to boost the response to sufficiently high titres to provide some protection. However, the antigenic distance of Omicron may mandate the development of vaccines against this strain.

There will then be a question of how to use these vaccines; vaccination with Omicron will likely give good protection against Omicron, but will not give good protection against other strains. It seems possible therefore that Omicron may cause a shift from the current monovalent vaccines containing Wuhan S to multivalent vaccines containing an antigen such as Wuhan or Alpha at the centre of the antigenic map and Omicron or other S genes at the extreme peripheries of the map, similar to the polyvalent strategies used in influenza vaccines.

In summary, we have presented data showing that the huge number of mutational changes present in Omicron lead to a substantial knock down of neutralizing capacity of immune serum and failure of mAb. This appears to lead to a fall in vaccine effectiveness, but it is unlikely that vaccines will completely fail and it is hoped that although vaccine breakthroughs J o u r n a l P r e -p r o o f will occur, protection from severe disease will be maintained, perhaps by T cells. It is likely that the vaccine induced T cell response to SARS-CoV-2 will be less affected than the antibody response. Third dose vaccine boosters substantially raise neutralization titres to Omicron and are, in countries such as the UK, the mainstay of the response to Omicron. Widespread vaccine breakthrough may mandate the production of a vaccine tailored to Omicron and failure of monoclonal antibodies may likewise lead to the generation of second generation mAbs targeting Omicron.

A question asked after the appearance of each new variant is whether SARS-CoV-2 has reached its limit for evolution. Analysing the mutations in Omicron shows that, except for S371L, all other mutations required only single-nucleotide changes. Two-nucleotide mutations and epistatic mutations are more difficult to reach, but open up vast untapped potentials for future variants. Global control measures are critical to avoid this.

The neutralization assays presented in this paper are performed in vitro, and do not fully quantify the antibody response in vivo where complement and antibody dependent cell mediated cytotoxicity may contribute to virus control. Evasion of the antibody response may allow reinfection with Omicron, but the role of the T cell response, not measured here, is likely to contribute to the control of infection and disease severity.

We are also grateful for support from Schmidt Futures, the Red Avenue Foundation and the (n=41), (H) 4 weeks, 6 months after the second dose, before the third and after the third dose of BNT162b2 (n=20). In A-E comparison is made with neutralization titres to Victoria, Alpha, Beta and Gamma and Delta previously reported in Supasa et al., 2021; Zhou et al., 2021; Dejnirattisai et al., 2021b; Liu et al., 2021b) , in G the data points for Victoria and Delta titres on BNT162b2 are taken from (Flaxman et al., 2021) . Geometric mean titres are shown above each column. The Wilcoxon matched-pairs signed rank test was used for the analysis and two-tailed P values were calculated. Victoria, Alpha, Beta, Gamma and Delta which have been previously reported Supasa et al., 2021; Zhou et al., 2021; Dejnirattisai et al., 2021b; Liu et al., 2021b) .

Neutralization titres are reported in Table S1 . Related to Figure S2 . respectively. These four Beta mAbs neutralise all the previous variants of concern as well as the early pandemic Wuhan strain. 

Resources, reagents and further information requirement should be forwarded to and will be responded by the Lead Contact, David I Stuart (dave@strubi.ox.ac.uk).

Reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

The coordinates and structure factors of the crystallographic complexes are available from the PDB with accession codes (see Table S2 ). Mabscape is available from https://github.com/helenginn/mabscape, https://snapcraft.io/mabscape. The data that support the findings of this study are available from the corresponding authors on request.

SARS-CoV-2/human/AUS/VIC01/2020 (Caly et al., 2020) , Alpha and Beta were provided by Public Health England, Gamma cultured from a throat swab from Brazil, Delta was a gift from J o u r n a l P r e -p r o o f (Folegatti et al., 2020) .

Data from vaccinated volunteers who received two vaccinations are included in this paper.

Vaccine doses were either 5 × 10 10 viral particles (standard dose; SD/SD cohort n=21) or half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort n=4). The interval between first and second dose was in the range of 8-14 weeks. Blood samples were collected and serum separated on the day of vaccination and on pre-specified days after vaccination e.g. 14 and 28 days after boost.

The neutralization potential of Ab was measured using a Focus Reduction Neutralization Test (FRNT), where the reduction in the number of the infected foci is compared to a negative control well without antibody. Briefly, serially diluted Ab or plasma was mixed with SARS-CoV-2 strain Victoria or P.1 and incubated for 1 hr at 37 °C. The mixtures were then transferred to 96-well, cell culture-treated, flat-bottom microplates containing confluent Vero cell monolayers in duplicate and incubated for a further 2 hrs followed by the addition of 1.5%

semi-solid carboxymethyl cellulose (CMC) overlay medium to each well to limit virus diffusion.

A focus forming assay was then performed by staining Vero cells with human anti-NP mAb J o u r n a l P r e -p r o o f (mAb206) followed by peroxidase-conjugated goat anti-human IgG (A0170; Sigma). Finally, the foci (infected cells) approximately 100 per well in the absence of antibodies, were visualized by adding TrueBlue Peroxidase Substrate. Virus-infected cell foci were counted on the classic AID EliSpot reader using AID ELISpot software. The percentage of focus reduction was calculated and IC50 was determined using the probit program from the SPSS package.

Cloning was done by using a restriction-free approach (Peleg and Unger, 2014 (Starr et al., 2020; Zahradnik et al., 2021a) .

Antigenic mapping of omicron was carried out through an extension of a previous algorithm . In short, coronavirus variants were assigned three-dimensional coordinates whereby the distance between two points indicates the base drop in neutralization titre. Each serum was assigned a strength parameter which provided a scalar offset to the logarithm of the neutralization titre. These parameters were refined to match predicted neutralization titres to observed values by taking an average of superimposed positions from 30 separate runs. The three-dimensional positions of the variants of concern: Victoria, Alpha, Beta, Gamma, Delta and Omicron were plotted for display.

Models of Omicron RBD and NTD were derived using AlphaFold 2.0.01 (Jumper et al., 2021) downloaded and installed on 11 th August 2021 in batch mode. For RBD predictions, 204 residues (P327-n529) were used as an input sequence while the NTD sequence input was from residues V1-S253. The max_release_date parameter was set to 28-11-2021 when the J o u r n a l P r e -p r o o f simulations were run such that template information was used for structure modelling. For all targets, five models were generated and all presets were kept the same.

Expression plasmids of wild-type and Omicron spike and RBD were constructed encoding for human codon-optimized sequences from wild-type SARS-CoV-2 (MN908947) and Omicron (EPI_ISL_6640917). Wild-type Spike and RBD plasmids were constructed as described before . Spike and RBD fragments of Omicron were custom synthesized by GeneArt (Thermo Fisher Scientific GENEART) and cloned into pHLsec and pNEO vectors, respectively, as previously described Supasa et al., 2021; . Both constructs were verified by Sanger sequencing after plasmid isolation using QIAGEN Miniprep kit (QIAGEN).

Protein expression and purification were conducted as described previously Zhou et al., 2020) . Briefly, plasmids encoding proteins were transiently expressed in HEK293T (ATCC CRL-11268) cells. The conditioned medium was concentrated using a

QuixStand benchtop system. His-tagged Omicron RBD were purified with a 5 mL HisTrap nickel column (GE Healthcare) and further polished using a Superdex 75 HiLoad 16/60 gel filtration column (GE Healthcare). Twin-strep tagged Omicron spike was purified with Strep-Tactin XT resin (IBA lifesciences).

~4mg of ACE2 was mixed with homemade His-tagged 3C protease and DTT (final concentration 1mM). After incubated at 4 °C for one day, the sample was flown through a 5 J o u r n a l P r e -p r o o f mL HisTrap nickel column (GE Healthcare). His-tagged proteins were removed by the nickel column and purified ACE2 was harvested and concentrated.

To purify full length IgG mAbs, supernatants of mAb expression were collected and filtered by a vacuum filter system and loaded on protein A/G beads over night at 4 °C. Beads were washed with PBS three times and 0.1 M glycine pH 2.7 was used to elute IgG. The eluate was neutralized with Tris-HCl pH 8 buffer to make the final pH=7. The IgG concentration was determined by spectro-photometry and buffered exchanged into PBS.

To express and purify Fabs 158 and EY6A, heavy chain and light chain expression plasmids of Fab were co-transfected into HEK293T cells by PEI. After cells cultured for 5 days at 37°C with 5% CO2, culture supernatant was harvested and filtered using a 0.22 mm polyethersulfone filter. Fab 158 was purified using Strep-Tactin XT resin (IBA lifesciences) and Fab EY6A was purified with Ni-NTA column (GE HealthCare) and a Superdex 75 HiLoad 16/60 gel filtration column (GE Healthcare).

AstraZeneca and Regeneron antibodies were provided by AstraZeneca, Vir, Lilly and Adagio antibodies were provided by Adagio. For the antibodies heavy and light chains of the indicated antibodies were transiently transfected into 293Y cells and antibody purified from supernatant on protein A. Fab fragments of 58 and beta-55 were digested from purified IgGs with papain using a Pierce Fab Preparation Kit (Thermo Fisher), following the manufacturer's protocol.

J o u r n a l P r e -p r o o f

The surface plasmon resonance experiments were performed using a Biacore T200 (GE Healthcare). All assays were performed with a running buffer of HBS-EP (Cytiva) at 25 °C.

To determine the binding kinetics between the SARS-CoV-2 RBDs and ACE2 / monoclonal antibody (mAb), a Protein A sensor chip (Cytiva) was used. ACE2-Fc or mAb was immobilised onto the sample flow cell of the sensor chip. The reference flow cell was left blank. RBD was injected over the two flow cells at a range of five concentrations prepared by serial twofold dilutions, at a flow rate of 30 μl min −1 using a single-cycle kinetics programme. Running buffer was also injected using the same programme for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

To determine the binding kinetics between the SARS-CoV-2 Spikes and ACE2, a CM5 sensor chip was used. The sensor chip was firstly activated by an injection of equal volume mix of EDC and NHS (Cytiva) at 20 uL/min for 300 s, followed by an injection of Spike sample at 20 ug/mL in 10 mM sodium acetate pH 5.0 (Cytiva) onto the sample flow cell of the sensor chip at 10 uL/min, and finally with an injection of 1.0 M Ethanolamine-HCl, pH 8.5 (Cytiva) at 20 uL/min for 180 s. The reference flow cell was left blank. ACE2 was injected over the two flow cells at a range of five concentrations prepared by serial twofold dilutions, at a flow rate of 30 μl min −1 using a single-cycle kinetics programme. Running buffer was also injected using the same programme for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

J o u r n a l P r e -p r o o f and scaled with the automated data processing program Xia2-dials (Winter, 2010; Winter et al., 2018) . 720° of data was collected from a crystal of Omicron-RBD/Beta-55/EY6A. 360° of data was collected for each of the Wuhan RBD/Beta-55/EY6A and Wuhan RDB/mAb-58/mAb-158 data sets.

Structures were determined by molecular replacement with PHASER (McCoy et al., 2007) .

VhVl and ChCl domains which have the most sequence similarity to previously determined SARS-CoV-2 RBD/Fab structures Dejnirattisai et al., 2021b; Huo et al., 2020; Liu et al., 2021a; Supasa et al., 2021; Zhou et al., 2021; Zhou et al., 2020) were used as search models for each of the current structure determination. Model rebuilding with COOT (Emsley et al., 2010) and refinement with Phenix (Liebschner et al., 2019) were used for all the structures. Data collection and structure refinement statistics are given in Table S2 .

Structural comparisons used SHP (Stuart et al., 1979) , residues forming the RBD/Fab interface were identified with PISA (Krissinel and Henrick, 2007) and figures were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).

Statistical analyses are reported in the results and figure legends. Neutralization was measured by FRNT. The percentage of focus reduction was calculated and IC50 (FRNT50) was determined using the probit program from the SPSS package. The Wilcoxon matched-pairs signed rank test was used for the analysis and two-tailed P values were calculated on geometric mean values.

Highlights  Large reduction in Omicron neutralization titres, ameliorated by 3 rd booster vaccine  Failure of many potent monoclonal antibodies to neutralize Omicron  Complex pattern of mutations balances ACE2 binding and antibody escape  Omicron RBD is structurally similar but the most antigenically distant variant eTOC A comprehensive analysis of sera from vaccinees, convalescent patients infected previously by multiple variants and potent monoclonal antibodies from early in the COVID-19 pandemic reveals a substantial overall reduction the ability to neutralize the SARS-CoV-2 Omicron variant, which a third vaccine dose seems to ameliorate. Structural analyses of the Omicron RBD suggest a selective pressure enabling the virus bind ACE2 with increased affinity that is offset by other changes in the receptor binding motif that facilitates immune escape.

Wuhan RBD was mixed with mAb-58 and mAb-158 Fabs, Wuhan or Omicron RBD was mixed with EY6A and beta-55 Fabs, in a 1:1:1 molar ratio

Crystals of Wuhan RBD/mAb-58/mAb-158 were formed in Hampton Research PEGRx condition 2-28, containing 0.1 M sodium citrate tribasic, pH 5.5 and 20% (w/v) PEG 4000

X-ray data collection, structure determination and refinement Light Source, UK. All data were collected as part of an automated queue system allowing unattended automated data collection

The threedimensional structure of foot-and-mouth disease virus at 2.9 A resolution

A time-and cost-efficient system for high-level protein production in mammalian cells

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies

Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies

Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion

Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift

SARS-CoV-2 evolved during advanced HIV disease immunosuppression has Beta-like escape of vaccine and Delta infection elicited immunity

Potent SARS-CoV-2 neutralizing antibodies directed against spike Nterminal domain target a single supersite

A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2

SARS-CoV-2 evolution in an immunocompromised host reveals shared neutralization escape mechanisms

Low-stringency selection of TEM1 for BLIP shows interface plasticity and selection for faster binders

Seroprevalence of SARS-CoV-2 Infection among Occupational Groups from the Bucaramanga Metropolitan Area, Colombia

The antigenic anatomy of SARS-CoV-2 receptor binding domain

Antibody evasion by the P.1 strain of SARS-CoV-2

Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail

Data, disease and diplomacy: GISAID's innovative contribution to global health

Features and development of Coot

Reactogenicity and immunogenicity after a late second dose or a third dose of ChAdOx1 nCoV-19 in the UK: a substudy of two randomised controlled trials (COV001 and COV002)

Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, singleblind, randomised controlled trial

Crystal structure of SARS-CoV-2 papain-like protease

Structure of the RNA-dependent RNA polymerase from COVID-19 virus

Defining variant-resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study

Breadth and function of antibody response to acute SARS-CoV-2 infection in humans

Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike

Highly accurate protein structure prediction with AlphaFold

Persistent SARS-CoV-2 infection and intra-host evolution in association with advanced HIV infection

SARS-CoV-2 evolution during treatment of chronic infection

Protein interfaces, surfaces and assemblies service PISA at European Bioinformatics Institute

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Human rhinovirus 3C protease: cloning and expression of an active form in Escherichia coli

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum

The Beta mAb response underscores the antigenic distance to other SARS-CoV-2 variants

An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies

The emergence and ongoing convergent evolution of the SARS-CoV-2 N501Y lineages

Phaser crystallographic software

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN

A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells

An oral SARS-CoV-2 M(pro) inhibitor clinical candidate for the treatment of COVID-19. Science, eabl4784

UCSF ChimeraX: Structure visualization for researchers, educators, and developers

Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody

A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology

Modular basis for potent SARS-CoV-2 neutralization by a prevalent VH1-2-derived antibody class

Deciphering key features in protein structures with the new ENDscript server

Structure of a human common cold virus and functional relationship to other picornaviruses

Human serum from SARS-CoV-2 vaccinated and COVID-19 patients shows reduced binding to the RBD of SARS-CoV-2 Omicron variant in comparison to the original Wuhan strain and the Beta and Delta variants

Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

Lentivirus-delivered stable gene silencing by RNAi in primary cells

Crystal structure of cat muscle pyruvate kinase at a resolution of 2.6 A

Emerging antibody-based therapeutics against SARS-CoV-2 during the global pandemic

Structure-based development of three-and four-antibody cocktails against SARS-CoV-2 via multiple mechanisms

Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera

Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein

Temporal dynamics of SARS-CoV-2 mutation accumulation within and across infected hosts

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion

A procedure for setting up high-throughput nanolitre crystallization experiments. I. Protocol design and validation

REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19

xia2: an expert system for macromolecular crystallography data reduction

DIALS: implementation and evaluation of a new integration package

Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19

Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation

Structural basis of a shared antibody response to SARS-CoV-2

A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets

SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution

SARS-CoV-2 convergent evolution as a guide to explore adaptive advantage

Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera

Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient

Potently neutralizing and protective human antibodies against SARS-CoV-2

This work was supported by the Chinese Academy of Medical Sciences (CAMS) Innovation