On the 24 th 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.

Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations: P.1 from Brazil, B.1.351 from South Africa and B.1.1.7 from the UK (12, 10 and 9 changes in the spike respectively). All have mutations in the ACE2 binding site with P.1 and B.1.351 having a virtually identical triplet: E484K, K417N/T and N501Y, which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine induced antibody responses than B.1.351 suggesting that changes outside the RBD impact neutralisation. Monoclonal antibody 222 neutralises all three variants despite interacting with two of the ACE2 binding site mutations, we explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.

SARS-CoV-2 has undergone progressive change with variants conferring advantage rapidly becoming dominant lineages e.g. B.1.617. With apparent increased transmissibility variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the UK. Here we study the ability of monoclonal antibodies, convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2 and complement this with structural analyses of Fab/RBD complexes and map the antigenic space of current variants. Neutralization of both viruses is reduced when compared with ancestral Wuhan related strains but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2 suggesting that individuals previously infected by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insight for immunisation policy with future variant vaccines in non-immune populations.

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Highlights d Original strain convalescent and vaccine sera show reduced B.1.1.7 neutralization d N501Y enhances RBD: ACE2 binding affinity d N501Y compromises neutralization by many antibodies with public V-region IGHV3-53 d No widespread escape by B.1.1.7 was observed

3Outer membrane proteins (OMPs) play important roles in Gram-negative bacteria, mitochondria and chloroplasts in nutrition transport, protein import, secretion, and other fundamental biological processes [1][2][3] . Dysfunction of mitochondria outer membrane proteins are linked to disorders such as diabetes, Parkinsons and other neurodegenerative diseases 4,5 . The OMPs are inserted and folded correctly into the outer membrane (OM) by the conserved OMP85 family proteins [6][7][8] , suggesting that similar insertion mechanisms may be used in Gram-negative bacteria, mitochondria and chloroplasts.In Gram-negative bacteria, OMPs are synthesized in the cytoplasm, and are transported across the inner membrane by SecYEG into the periplasm 8,9 . The seventeen kilodalton (kDa) protein (Skp) and the survival factor A (SurA) chaperones escort the unfolded OMPs across the periplasm to the β-barrel assembly machinery (BAM), which is responsible for insertion and assembly of OMPs into the OM 10-12 . InEscherichia coli, the BAM complex consists of BamA and four lipoprotein subunits, BamB, BamC, BamD and BamE. BamA is comprised of five N-terminal polypeptide transport-associated (POTRA) domains and a C-terminal OMP transmembrane barrel, while the four lipoproteins are affixed to the membrane by N-terminal lipid-modified cysteines. Of these subunits, BamA and BamD are essential 3,6 . One copy of each of these five proteins is required to form the BAM complex with an approximate molecular weight of 200 kDa (Extended Data Fig. 1). In vitro reconstitution of the E.coli BAM complex and functional assays showed that all five subunits are required to obtain the maximum activity of BAM [13][14][15][16] . Furthermore, comparison of the two complexes reveals that the periplasmic units are rotated with respect to the barrel, which appears to be linked to significant conformational changes in the β-strands β1C-β6C of the barrel. Taken together this suggests a novel insertion mechanism whereby rotation of the BAM periplasmic ring promotes insertion of OMPs into the OM. To our knowledge, this is the first reported crystal structure of an intramembrane barrel with a lateral-open conformation.Unique architecture of two E. coli BAM complexes X-ray diffraction data of selenomethionine labelled crystals were collected to 3.9Ångström (Å) resolution and the BAM structure was determined by singlewavelength anomalous dispersion (SAD) and manual molecular replacement (Methods, Extended Data Table 1). The first structure contained four proteins: BamA, BamC, BamD and BamE (Fig. 1a-c), with the electron density and crystal packing indicating that the BamB is absent in the complex. This was confirmed by SDS-PAGE analysis of the crystals (Extended Data Fig. 1 and Supplementary Data Fig. S1). In this model, BamA, BamC, BamD and BamE contain residues E22-I806, C25-K344, E26-S243, and C20-E110, respectively. The machinery is approximately 115 Å in length, 84 Å in width and 132 Å in height (Fig. 1a). 5The architecture of BamACDE resembles a top hat with a...

Highlights d Map 377 mAbs: 19 of 80 recognizing the RBD are potent neutralizers; 1 potent NTD binder d 19 Fab-antigen complex structures; 80 mAbs mapped on RBD and clustered into 5 epitopes d Most potent mAbs are ACE2 blockers, neutralize with few ACE2s, some Fabs glycosylated d mAbs reveal unique examples of NTD binding, RBD binding mode, and LC optimization

ARS-CoV-2 was first detected in December 2019, leading to a pandemic with an estimated 5-6% mortality rate 1. Akin to SARS-CoV-1, the causative agent of the 2003 SARS outbreak, this is an enveloped betacoronavirus with protrusions of large trimeric 'spike' proteins. Receptor binding domains (RBDs) located at the tips of these spikes facilitate host cell entry via interaction with angiotensin-converting enzyme 2 (ACE2) 2. Spikes are type I transmembrane glycoproteins, formed from a single polypeptide, which transitions into a post-fusion state via cleavage into S1 (N-terminal) and S2 (C-terminal) chains following receptor binding or trypsin treatment 3. In the pre-fusion state, the apical RBD (~22 kDa) is folded down, enshrouded by the N-terminal domain (NTD) of the spike so that the receptor binding site is inaccessible until, it is assumed, an RBD stochastically swings upwards to present the ACE2 binding site 4-7. ACE2 interaction locks the RBD in the 'up' conformation, which drives conversion to the post-fusion form where the S2 subunit engages the host membrane while dispensing with S1 4,5. Neutralizing human monoclonal antibodies (mAbs) that recognize the ACE2 receptor binding site for SARS-CoV-1 and SARS-CoV-2 are generally not cross-reactive between the two viruses and are susceptible to escape mutation 8-12. Indeed, a natural mutation (Y495N) has already been identified at this site (GISAID 13 : accession ID: EPI_ISL_429783 Wienecke-Baldacchino et al.). By contrast, the CR3022 antibody (derived from a SARS-CoV-1-infected patient) cross-reacts strongly with SARS-CoV-2 (see Methods and Fig. 1) and has been shown to recognize a cryptic, conserved footprint on the RBD distinct from the binding epitope of