Characterization of the enhanced infectivity and antibody evasion of Omicron BA.2.75

Abstract

Recently emerged SARS-CoV-2 Omicron subvariant, BA.2.75, displayed a growth advantage over circulating BA.2.38, BA.2.76, and BA.5 in India. However, the underlying mechanisms for enhanced infectivity, especially compared with BA.5, remain unclear. Here, we show that BA.2.75 exhibits substantially higher affinity for host receptor angiotensin-converting enzyme 2 (ACE2) than BA.5 and other variants. Structural analyses of BA.2.75 spike shows its decreased thermostability and increased frequency of the receptor binding domain (RBD) in the "up" conformation under acidic conditions, suggesting enhanced low-pH-endosomal cell entry. Relative to BA.4/BA.5, BA.2.75 exhibits reduced evasion of humoral immunity from BA.1/BA.2 breakthrough-infection convalescent plasma but greater evasion of Delta breakthrough-infection convalescent plasma. BA.5 breakthrough-infection plasma also exhibits weaker neutralization against BA.2.75 than BA.5, mainly due to BA.2.75's distinct neutralizing antibody (NAb) escape pattern. Antibody therapeutics Evusheld and Bebtelovimab remain effective against BA.2.75. These results suggest BA.2.75 may prevail after BA.4/BA.5, and its increased receptor-binding capability could support further immune-evasive mutations.

Keywords: BA.2.75; COVID-19; Omicron; SARS-CoV-2; cryo-EM structure; immune evasion; neutralizing antibody; spike glycoprotein.

Graphical abstract

Figure 1

BA.2.75 and BA.2.76 displayed growth advantage in India (A) Main mutations on the spike glycoprotein appearing in SARS-CoV-2 Omicron sublineages. (B) Phylogenetic tree of existing SARS-CoV-2 variants. Color scales indicate number of mutations on the spike. (C) Lineage distribution of recent sequences from India by time. BA.2.75 and BA.2.76 is growing rapidly in India, showing advantage compared with other lineages.

Figure 2

BA.2.75 exhibited enhanced human ACE2 binding (A) Position distribution of important amino acids in BA.2.75 RBD. The mutated residues relative to BA.2 RBD are marked as orange globules. RBM is colored in red. (B) Binding affinities of RBDs of BA.2, BA.4/5, and BA.2.75 subvariants to hACE2 measured by SPR. (C) Overall structure of the BA.2.75 S-trimer in complex with hACE2. Three copies of S monomer are colored in yellow, cyan, and magenta, respectively. The hACE2 molecules bound to RBD are colored in orange. (D) Changes at the interfaces between BA.2.75 RBD (left) and BA.2 RBD (PDB: 7ZF7, right) with hACE2. Key mutated residues are shown as sticks and hydrogen bonds are shown as yellow dash lines. (E) Binding affinity of hACE2 with BA.2.75 RBD with single substitution Q493R, S446G, and K460N measured by SPR. (F) Binding affinity of hACE2 with BA.2+N460K RBD measured by SPR. See also Figure S1.

Figure 3

Structural characteristics of BA.2.75 spike glycoprotein (A) Surface representation for structures of S-trimer of BA.2.75 at neutral (pH = 7.4) and acidic conditions (pH = 5.5); three protomers were colored in yellow, light blue, and pink, respectively, and N-glycans were highlighted in deep blue. (B) Buried surface areas between two neighboring protomers, S2-subunits, S1-subunits, or RBD domains. (C) Structural organization of three S1-subunits and RBDs from the neutral (gray) and acidic BA.2.75 S-trimer (yellow, light blue, and pink). Top view of S2 subunit (left), top view of the RBD (upper right), and central section of RBD (bottom right) show the inter-subunit contacts of the BA.2.75 S-trimers in different pH. (D) Superimposition of the neutral BA.2.75 S-trimer structure (gray) onto the structure of the acidic BA.2.75 S-trimer (RBD, yellow; NTD, hot pink); Structural rotations and shifts between these two structures were marked by green lines and arrows. (E) Structural alterations in the 630 loop (residues 617–644, light blue) and FPPR (residues 823–862, yellow) of the three protomers (a, b, and c) from these two S-trimers were shown. Dashed lines indicate gaps in the chain trace (disordered loops). (F) Thermal stability analysis of BA.1 (green), BA.2 (blue), BA.2.12.1 (black), BA.2.75 (red), and BA.5 (yellow) S-trimers at neutral and acidic pHs. See also Figure S2.

Figure 4

Structural features of BA.2.75 spike RBD and NTD (A) Structural comparisons of RBDs of BA.2.75, BA.2, and BA.1. The newly established interaction of BA.2.75 (pink) with respect to BA.2 (gray) on the RBD is shown on the left. Salt bridges formed between D420 and K460 and π-π stack formed between H339 and F371 in BA.2.75 RBD are highlighted. The distances between α1 and α2 helices on RBD are also marked. A diagram presentation of N343 glycan conformational differences among BA.1 (gray), BA.2 (blue), and BA.2.75 (pink) is shown on the right. (B) The stability landscapes of BA.2.75 and BA.2 RBD. The cartoons of BA.2.75 and BA.2 RBD are colored by root mean square fluctuation (RMSF) calculated from the last 2 ns of the MD stimulations. Residues 339, 446, 460, and 493 are shown as red spheres. (C) Thermal stability measurements of the RBD from BA.1 (green), BA.2 (blue), BA.5 (orange), BA.2.12.1 (black), and BA.2.75 (red) at pH 7.4. (D) Entropy of SARS-CoV-2 NTD variants among circulating isolates. The residues with higher entropy are highlighted by dark red background. The dominant mutations on SARS-CoV-2 NTD and the mutations on BA.2.75 NTD are labeled in black and red, respectively. (E) The heatmap for circulating variants with mutations on the NTD. Mutation frequency for each residue is calculated based on the datasets from Global Initiative on Sharing All Influenza Data (GISAID). (F) Cartoon representation of NTD colored by mutation frequency as same as (E). Five mutations in the BA.2.75 NTD are displayed as blue balls and labeled. The secondary structure these mutations locate are also labeled. See also Figure S3.

Figure 5

BA.2.38, BA.2.75, and BA.2.76 showed distinct antibody evasion (A) Half neutralization titers (NT50s) against SARS-CoV-2 D614G and Omicron variants pseudoviruses by plasma samples from individuals who received 3 doses CoronaVac (n = 40), 3 doses CoronaVac followed by BA.1 infection (n = 50), 3 doses CoronaVac followed by BA.2 infection (n = 39), 2 doses CoronaVac followed by Delta infection (n = 16), or 3 doses CoronaVac followed by BA.5 (n = 8) infection. Geometric mean titers (GMTs) are annotated above each group. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. p values are calculated using two-tailed Wilcoxon signed-rank test of paired samples. Pair-wise fold changes were calculated using the NT50 against the strain on the left divided by that against the strain on the right. (B) Neutralizing activities against SARS-CoV-2 D614G, Omicron variants, and SARS-CoV-1 pseudovirus of therapeutic neutralizing antibodies. Background colors indicate neutralization levels. Green, IC50 < 30 ng/mL; white, 30 ng/mL < IC50 < 1,000 ng/mL; red, IC50 > 1,000 ng/mL. ^∗IC50 > 10,000 ng/mL. (C) Correlation plots of binding affinities and neutralizing activities (IC90) of S309 against BA.1, BA,2, BA.2.12.1, BA.4/5, and BA.2.75. (D) Local conformational alterations in the BA.2.75 RBD upon S309 binding. The N343 glycan of the apo BA.2.75 RBD (gray) and S309-bounded BA.2.75 RBD (yellow) are shown as sticks and the distances between α1 and α2 helices from two configurations are also labeled. (E) Interaction details of the BA.1 RBD (left) and BA.2.75 RBD (right) in complex with S309. Hydrogen bonds and hydrophobic patches are presented as yellow dashed lines and gray surfaces, respectively. The light chain and heavy chain of S309 are colored in pink and cyan, respectively. See also Figures S4 and S5.

Figure 6

Evasion of NAbs targeting various RBD epitopes by BA.2.38, BA.2.75, and BA.2.76 Neutralizing activities against SARS-CoV-2 Omicron variants of NAbs in (A) group A (n = 11 isolated from SARS-CoV-2^WT convalescents or vaccinees; n = 18 from post-vaccination BA.1 convalescents), (B) group D1 (n = 17, 18, respectively), (C) group D2 (n = 18, 17, respectively), (D) group B (n = 18), and (E) group E1 (n = 18). Deep mutational scanning (DMS) profiles of antibodies in groups A, D1, D2, and E1 are projected onto RBD structure to show interacting hotspots of each group. Color shades indicate escape scores of RBD residues. Interface structural models of representative antibodies in groups A, D1, D2, and E1, in complex of RBD, show potential escaping mechanism of BA.2.75 and BA.2.76. Geometric mean of IC50 fold changes compared with BA.2 are annotated above the bars. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. p values are calculated using two-tailed Wilcoxon signed-rank test of paired samples.

Figure 7

BA.2.75 and BA.2.76 evolve mutations to escape NTD-targeting NAbs (A) Heatmap of pseudo-typed virus neutralization by antibodies recognized NTD. (B) Cryo-EM structures of the BA.2.75 S-trimer in complex with XG2v024 Fab (top). Structures are shown as surface. The three subunits of S protein are colored in yellow, cyan, and magenta, respectively. The heavy chain and light chain are colored in hot pink and orange, respectively. Interactions between the XG2v024 and BA.2.75 NTD (bottom). The CDRs of the XGv024 that interact with BA.2.75 NTD are displayed as thick tubes over the magenta surface of the NTD. The XG2v024 epitope is shown as a cartoon representation over the surface of the XG2v024 Fab. (C) XG2v024 neutralizing mechanism. The BA.2.75 S-trimer and XG2v024 Fab are shown as surface. The color scheme remains unchanged from the previous panels. Steric clash is marked as black stars. (D) Interactions details of BA.2.75 NTD in complex with XG2v024 Fab. The hydrophobic (top) and hydrophilic (bottom) interactions are shown, respectively. Hydrogen bonds and hydrophobic patches are presented as yellow dashed lines and gray surfaces, respectively. (E) Structural landscapes of the four classes of NTD NAbs. Antigenic patches recognized by four types of NAbs are outlined in the assigned color scheme. Five mutations in the BA.2.75 NTD are labeled. (F) Structure-based antigenic clustering of SARS-CoV-2 NTD Nabs (left). A total of 38 NTD NAbs with available structures are classified into four clusters (α, β, γ, and δ). Surface representative model of four types of NAbs bound to the NTD (right). Fab fragments of four representative antibody are shown in different colors and the NTD is colored in gray. (G) Analysis of sequence conservation of XG2v024 epitope. The logo plot represents the conservation of XG2v024 epitope residues from 26 SARS-CoV-2 lineages. See also Figures S6 and S7.