key: cord-0273198-kh0r9prr
authors: Wang, Qian; Guo, Yicheng; Iketani, Sho; Li, Zhiteng; Mohri, Hiroshi; Wang, Maple; Yu, Jian; Bowen, Anthony D.; Chang, Jennifer Y.; Shah, Jayesh G.; Nguyen, Nadia; Meyers, Kathrine; Yin, Michael T.; Sobieszczyk, Magdalena E.; Sheng, Zizhang; Huang, Yaoxing; Liu, Lihong; Ho, David D.
title: SARS-CoV-2 Omicron BA.2.12.1, BA.4, and BA.5 subvariants evolved to extend antibody evasion
date: 2022-05-26
journal: bioRxiv
DOI: 10.1101/2022.05.26.493517
sha: 58499a2cc5f026d75cbb5ba56319b6153092da2e
doc_id: 273198
cord_uid: kh0r9prr

The Omicron subvariant BA.2 accounts for a large majority of the SARS-CoV-2 infection worldwide today1. However, its recent descendants BA.2.12.1 and BA.4/5 have surged dramatically to become dominant in the United States and South Africa, respectively2,3. That these novel Omicron subvariants carry additional mutations in their spike proteins raises concerns that they may further evade neutralizing antibodies, thereby further compromising the efficacy of our COVID-19 vaccines and therapeutic monoclonals. We now report findings from a systematic antigenic analysis of these surging Omicron subvariants. BA.2.12.1 is only modestly (1.8-fold) more resistant to sera from vaccinated and boosted individuals than BA.2. On the other hand, BA.4/5 is substantially (4.2-fold) more resistant and thus more likely to lead to vaccine breakthrough infections. Mutation at spike residue L452 found in both BA.2.12.1 and BA.4/5 facilitates escape from some antibodies directed to the so-called Class 2 and Class 3 regions of the receptor-binding domain (RBD)4. The F486V mutation found in BA.4/5 facilitates escape from certain Class 1 and Class 2 antibodies to the RBD but compromises the spike affinity for the cellular receptor ACE2. The R493Q reversion mutation, however, restores receptor affinity and consequently the fitness of BA.4/5. Among therapeutic antibodies authorized for clinical use, only bebtelovimab (LY-COV1404) retains full potency against both BA.2.12.1 and BA.4/5. The Omicron lineage of SARS-CoV-2 continues to evolve, successively yielding subvariants that are not only more transmissible but also more evasive to antibodies.

in the northeast region that includes New York (Fig. 1a) , where it now accounts for over 70% of 35 all new SARS-CoV-2 infections 3 . BA.4 and BA.5 emerged in South Africa in January and rapidly 36 became dominant there with a combined frequency of over 88% 1 . These new Omicron subvariants 37 have been detected worldwide, albeit at low levels presently. However, their growth trajectories 38 in the U.S. and South Africa indicate a significant transmission advantage that will likely result in 39

Affinity to human ACE2 94

Epidemiological data clearly indicate that both BA.2.12.1 and BA.4/5 are very transmissible (Fig.  95   1a) ; however, the additional mutations at the top of RBD (Fig. 2c ) of these subvariants raises the 96 possibility of a significant loss of affinity for the viral receptor, human ACE2 (hACE2), as has 97 been reported by one group 22 . We therefore measured the binding affinity of purified spike 98 proteins of D614G and major Omicron subvariants (Extended Data Fig. 7) to dimeric hACE2 using 99 surface plasmon resonance (SPR). The spike proteins of the Omicron subvariants exhibited similar 100 binding affinities to hACE2, with KD values ranging from 1.66 nM for BA.4/5 to 2.36 nM for 101 BA.2.12.1 to 2.79 nM for BA.1.1 (Fig. 3a) . Impressively, despite having >17 mutations in the 102 RBD to escape antibody neutralization, BA.2.12.1 and BA.4/5 also evolved concurrently to gain 103 a slightly higher affinity for the receptor than an ancestral SARS-CoV-2, D614G (KD 5.20 nM). 104 105 To support the findings by SPR and to probe the role of point mutations in hACE2 binding, we 106 tested BA.2, BA.2.12.1, and BA.4/5 pseudoviruses, as well as pseudoviruses containing key 107 mutations, to neutralization by dimeric hACE2 in vitro. The IC50 values were lower for BA.4/5 108 and BA.2.12.1 than that of BA.2 (Fig. 3b) , again indicating that these two emerging Omicron 109 subvariants have not lost receptor affinity. Our results also showed that the F486V mutation 110 compromised receptor affinity, as previously reported 23 , while the R493Q reversion mutation 111 improved receptor affinity. To structurally interpret these results, we modeled F486V and R493Q 112 mutations based on the crystal structure of BA.1-RBD-hACE2 complex 24 overlaid with ligand-113 free BA.2 RBD (PDB: 7U0N and 7UB0). This analysis found that both R493 and F486 are 114 conformationally similar between BA.1 and BA.2, and F486V led to a loss of interaction with a 115 hydrophobic pocket in hACE2 (Fig. 3c) Extended Data Fig. 9 ). S704L, a mutation close to the S1/S2 cleavage site, did not appreciably 147 alter the serum neutralization titers against BA.2. For "boosted" serum samples, the impact of 148 each point mutant on neutralization resistance was quantified and summarized in Fig. 4b . 149

Using these serum neutralization results, we then constructed a graphic display to map antigenic 151 distances among D614G, various Omicron subvariants, and individual point mutants for just the 152 "boosted" samples to avoid confounding effects from differences in clinical histories in the other 153 cohorts. The resultant antigenic cartography (Fig. 4d) higher affinity for the receptor, contrary to suggestions that it might be less fit 22 . 177 confers antibody sensitivity (Fig. 4b) . Moreover, the data generated using SARS-CoV-2-181 neutralizing mAbs suggest that a mutation at L452 allows escape from Class 2 and Class 3 RBD 182 antibodies and that the F486V mutation mediates escape from Class 1 and Class 2 RBD antibodies 183 

Global initiative on sharing all influenza data -from vision to 237 reality

Omicron sub-lineages BA.4/BA.5 escape BA.1 infection elicited neutralizing 239 immunity. medRxiv

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies

Studies in humanized mice and convalescent humans yield a SARS-CoV-2 245 antibody cocktail

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The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in 249 nonhuman primates

A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2

Human neutralizing antibodies elicited by SARS-CoV-2 infection

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

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants

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An elite broadly neutralizing antibody protects SARS-CoV-2 Omicron variant 267 challenge. bioRxiv

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BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection. bioRxiv

Structural Basis for Human Receptor Recognition by SARS-CoV-2 Omicron Variant Methods 305

The SARS-CoV-2 spike structure (6ZGE) used for displaying epitope footprints and mutations 399 within the emerging variants was downloaded from the Protein Data Bank (PDB). Epitope residues 400 were identified using PISA 31 with default parameters, and the RBD residues with non-zero buried 401 accessible surface area were considered epitope residues. For each residue within the RBD, the 402 frequency of antibody recognition was calculated as the number of contact antibodies 32 . The 403 structures of antibody-spike complexes for modeling were also obtained from PDB (7L5B (2-15), 404 6XDG (REGN10933), and 7KMG (LY-CoV555)). PyMOL v.2.3.2 was used to perform 405 mutagenesis and to generate structural plots (Schrödinger). 406

The antigenic distances between SARS-CoV-2 variants were approximated by incorporating the 409 neutralization potency of each serum sample into a previously described antigenic cartography 410 approach 33 . The map was generated by the Racmacs package (https://acorg.github.io/Racmacs/, 411 version 1.1.4) in R with the optimization steps set to 2000, and with the minimum column basis 412 parameter set to "none". 413