Fig 1: The functionality of anti-S2 mAbs on inhibition of membrane fusion and virus infection. (A) For performing the S protein-mediated syncytium formation assay, ZIKV-A1 (isotype control antibody), S2-10H, S2-4D, S2-5D, S2-8D, or S2-8A (100 µg/mL for each) was preincubated with S protein-transfected 293T cells individually, before being added to the EGFP-transfected ACE2-293T cells. After 16 h of coculture, the syncytium formation was observed by fluorescence microscopy. (B) For performing the three biological replicates (n = 3) of plaque reduction assay, the indicated antibody (100 µg/mL) was incubated with the SARS-CoV-2 wild-type strain in the presence of 8 µg/mL TPCK-trypsin of DMEM for 1 h at 37°C. Antibody-virus mixtures (200 µL/well) were subsequently added to the Vero E6 cell monolayers for one additional hour in 24-well plates. Five to 7 days later, cells were fixed with formaldehyde and stained with crystal violet. (C–E) The 50% of neutralization (EC50) of SARS-CoV-2 infection by mAbs S2-4D, S2-5D, and S2-8D were determined by using a series of diluted MAb solutions (80, 40, 20, 10, 5 µg/mL) in the plaque reduction assay. Data are presented as means ± SD of three biological replicates (n =3), and further graphed by linear regression.
Fig 2: The virus neutralizing activity and antigenic specificity of MAb S2-4A. (A) The S protein-mediated syncytium formation assay was performed to analyze the neutralizing activity of MAb S2-4A. The formation of the syncytia by coculturing the EGFP-transfected ACE2-293T cells with the S protein-transfected 293T cells was markedly inhibited in the presence of S2-4A (100 µg/mL). (B) The MAb S2-4A (100 µg/mL) was incubated with the SARS-CoV-2 wild-type strain. and the plaque reduction assay was applied to evaluate the neutralizing activity. Data are three biological replicates (n = 3). (C) The EC50 of MAb S2-4A against SARS-CoV-2 was determined by using a series of MAb S2-4A (100, 50, 25, 12, 5 µg/mL) in the plaque reduction assay. (D) The binding of MAb S2-4A to S2 truncation fragments #4, #10, #11, and #12 was determined by WB analysis. (E) The sfGFP-S(1042-1167) mutants with a series of individual alanine substitution at residues 1137–1164 were analyzed on SDS-PAGE and then subjected to WB analysis with MAb S2-4A to identify the critical antigenic determinants. (F) The S2-4A (100 µg/mL) was preincubated with the SARS-CoV-2 wild-type strain (white bar), Alpha (gray bar), Epsilon (blue bars), Delta (yellow bars), and Gamma (black bars) variants and then subjected to the plaque reduction assay. The inhibition of the plaque formation by MAb S2-4A was calculated by comparing to the experimental results in the absence of antibodies. Data are presented as means ± SD of three biological replicates (n = 3). One-way ANOVA was used for statistical analysis. *, P = 0.05; **, P = 0.01; ***, P = 0.001. (G–J) For determination of the EC50 values of S2-4A against SARS-CoV-2 Alpha, Epsilon, Delta, and Gamma variants, a series of diluted MAb S2-4A solutions (80, 40, 20, and 10 µg/mL) were utilized in the plaque reduction assay. Data are presented as means ± SD of three biological replicates (n =3), and further graphed by linear regression. (K) The EC50 values of S2-4A against different SARS-CoV-2 variants.
Fig 3: Functional and structural basis of class II antibody binding, neutralization, and escape.(A) Lentiviruses pseudotyped with SARS-CoV-2 spike proteins from D614G or D614G plus the indicated point substitutions found within the B.1.1.529 spike were incubated with serial dilutions of the indicated antibodies, and IC50 and IC80 values were determined on 293T-ACE2 cells. Ranges are indicated with white (>10,000 ng/ml), light blue (>1000 to =10,000 ng/ml), yellow (>100 to =1000 ng/ml), orange (>50 to =100 ng/ml), red (>10 to =50 ng/ml), maroon (>1 to =10 ng/ml), and purple (=1 ng/ml). (B) Cryo-EM structure of class II antibody A19-46.1 Fab in complex with the B.1.1.529 spike. Overall density map is shown to the left, with protomers in light green, gray, and light cyan. Two A19-46.1 Fabs bound to the RBD in the up conformation are shown in orange and slate. Structure of the RBD and A19-46.1 after local focused refinement is shown to the right in cartoon representation. The heavy-chain CDRs are in brown, pink, and orange for CDR H1, CDR H2, and CDR H3, respectively. The light chain CDRs are in marine purple blue, marine blue, and blue for CDR L1, CDR L2, and CDR L3, respectively. The contour level of the cryo-EM map is 4.0s. (C) Interaction between A19-46.1 and RBD. (Left) CDR H3 and all light-chain CDRs that are involved in binding of RBD. Epitope of A19-46.1 is shown in orange on the green B.1.1.529 RBD surface, with amino acid substitutions in red. (Right) S446, A484, and R493 are located at the edge of the epitope of Fab A19-46.1. RBD residues are labeled with italicized font. (D) Binding of A19-46.1 to RBD prevents binding of the ACE2 receptor. ACE2 and A19-46.1 are shown in cartoon representation. (E) Comparison of binding modes to RBD for antibody A19-46.1 and LY-CoV555. (Left and inset) Even though both antibodies target similar regions on RBD, different approaching angles caused a clash between LY-CoV555 CDR H3 and B.1.1.529 substitution R493. (Right) B.1.1.529 substitutions involved in binding of A19-46.1 are only at the edge of its epitope, whereas both R493 and A484 locate in the middle of LY-CoV555 epitope. L452R substitution that eliminates A19-46.1 and LY-CoV555 binding in other SARS-CoV-2 variants is in blue.
Fig 4: Cryo-EM structure of the SARS-CoV-2 B.1.1.529 (Omicron) spike.(A) Cryo-EM map of the SARS-CoV-2 B.1.1.529 spike. Reconstruction density map at 3.29 Å resolution is shown with side and top views. Protomers are colored light green, wheat, and light blue. The contour level of cryo-EM map is 4.0s. (B) B.1.1.529 amino acid substitutions introduced interprotomer interactions. Substitutions in one of the protomers are shown as red spheres. Examples of interprotomer interactions introduced by B.1.1.529 substitutions are highlighted in the box with zoom-in views to the side. Amino acid substitutions are described as a percentage of the domain surface (surface) or as a percentage of the sequence (seq). (C) The NTD supersite of vulnerability is shown in semitransparent surface along with a green backbone ribbon. Amino acid substitutions, deletions, and insertions are in red. (D) The 15 amino acid substitutions, clustered on the rim of RBD, changed 16% of the RBD surface area (left) and increased electropositivity of the ACE2-binding site (right). Amino acid substitutions are shown as red sticks. The ACE2-binding site on the electrostatic potential surface are marked as magenta lines. (E) Mapping B.1.1.529 RBD substitutions on the epitopes of Barnes class I to IV antibodies. The locations of the substitutions are shown in red on the surface. Those that may potentially affect the activity of antibodies in each class are labeled with their residue numbers. Class I footprint is defined by epitopes of CB6 and B1-182.1; class II footprint is defined by epitopes of A19-46.1 and LY-CoV555; class III footprint is defined by epitopes of A19-61.1, COV2-2130, LY-CoV1404 and S309; and class IV footprint is defined by epitopes of DH1047 and S304. Class I and II antibodies primarily target the ACE2 binding site, whereas the epitopes of class III and IV antibodies do not. Class II and III epitopes allow binding to WA-1 when RBD is in the up or down conformation, although the distinction between class I and II is more fluid, particularly with new variants that alter the accessibility of epitopes relative to WA-1. In addition, some antibodies, such as A19-46.1, can bind fully up intermediate states between up and down but cannot bind the fully down state. We therefore classified primarily by binding region.
Fig 5: Functional and structural basis of class I antibody neutralization and mechanistic basis of retained potency against B.1.1.529 VOC.(A) Lentiviruses pseudotyped with SARS-CoV-2 spike proteins from D614G or D614G plus the indicated point substitutions found within the B.1.1.529 spike were incubated with serial dilutions of the indicated antibodies, and IC50 and IC80 values were determined on 293T-ACE2 cells. Ranges are indicated with white (>10,000 ng/ml), light blue (>1000 to =10,000 ng/ml), yellow (>100 to =1000 ng/ml), orange (>50 to =100 ng/ml), red (>10 to =50 ng/ml), maroon (>1 to =10 ng/ml), and purple (=1 ng/ml). (B) Mapping of B.1.1.529 amino acid substitutions at the epitope of class I antibody CB6. RBD-bound CB6 was docked onto the B.1.1.529 spike structure. B.1.1.529 amino acid substitutions incompatible with CB6 binding were identified and labeled. The K417N substitution caused a clash in the center of the paratope. B.1.1.529 RBD is shown in green cartoon, with amino acid substitutions as red sticks. CB6 is shown in surface representation, with heavy and light chains in yellow and slate, respectively. (C) Docking of RBD-bound VH1-58–derived class I antibody B1-182.1 onto the B.1.1.529 spike structure identified four substitutions with potential steric hindrance. B1-182 is shown in surface representation, with heavy and light chains colored olive and light blue, respectively. B.1.1.529 amino acid substitutions that may affect binding of VH1-58 antibodies were labeled. (D) Structural basis for effective neutralization of the B.1.1.529 VOC by VH1-58–derived antibodies. Even though VH1-58 antibodies—such as the S2E12, COV2-2196, A23-58.1, and B1-182.1—share high-sequence homology (top right), their neutralization potency against B.1.1.529 varies. Structural analysis indicated that CDR H3 residue 100C, located at the interface formed between RBD and antibody heavy and light chains, may determine their potency against B.1.1.529 (left). Size of this residue correlated with neutralization potency with two-tailed P = 0.046 (bottom right).
Supplier Page from Sino Biological, Inc. for Human ACE2 / Angiotensin-Converting Enzyme 2 Gene ORF cDNA clone expression plasmid, C-His tag