Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge during the global pandemic and may facilitate escape from current antibody therapies and vaccine protection. Here we showed that the South African variant B.1.351 was the most resistant to current monoclonal antibodies and convalescent plasma from coronavirus disease 2019 (COVID-19)-infected individuals, followed by the Brazilian variant P.1 and the United Kingdom variant B.1.1.7. This resistance hierarchy corresponded with Y144del and 242-244del mutations in the N-terminal domain and K417N/T, E484K, and N501Y mutations in the receptor-binding domain (RBD) of SARS-CoV-2. Crystal structure analysis of the B.1.351 triple mutant (417N-484K-501Y) RBD complexed with the monoclonal antibody P2C-1F11 revealed the molecular basis for antibody neutralization and escape. B.1.351 and P.1 also acquired the ability to use mouse and mink ACE2 receptors for entry. Our results demonstrate major antigenic shifts and potential broadening of the host range for B.1.351 and P.1 variants, which poses serious challenges to current antibody therapies and vaccine protection.

Keywords: SARS-CoV-2; antibody; immune escape; neutralization; variant of concern.

Graphical abstract

Figure 1

SARS-CoV-2 variants and single amino acid mutations reduce neutralization and binding of mAbs (A) Mutant residues along the S protein identified in SARS-CoV-2 variants B.1.1.7, B.1.351, and P.1. Mutant residues in each variant are indicated by black vertical lines. Those highlighted in yellow represent the mutant residues studied here. Nine mutant residues are underlined that were not identified in the three variants but included in the study because of their high representation in the GISAID database. SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain; RBM, receptor binding motif; SD, subdomain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain; CP, cytoplasmic domain. (B) The binding mode to and footprint of ACE2 and four classes of antibodies on the RBD. ACE2 is colored in green, RBD in cyan, and representative antibodies in four distinct colors. The three mutation residues (K417, E484, and N501) critical for binding to ACE2 and antibodies are shown in red. (C and D) The fold changes in neutralizing activity, measured by half-maximal inhibitory concentration (IC₅₀) (C), and in binding activity, measured by mean fluorescence intensity (MFI), relative to that of WT D614G. “−” indicates increased resistance and “+” increased sensitivity. IC₅₀ or MFI values highlighted in red indicate that resistance increased at least 3-fold; in blue, sensitivity increased at least threefold; and in white, resistance or sensitivity increased less than 3-fold. BDL (below the detection limit) indicates that the highest concentration of mAbs failed to reach 50% neutralization. Results were calculated from three independent experiments performed in technical duplicates. See also Figures S1 and S2.

Figure 2

Structural basis for mAb neutralization and escape (A) P2C-1F11/RBD-3M crystal structure superimposed onto the P2C-1F11/RBD crystal structure (PDB: 7CDI). P2C-1F11 is colored magenta and RBD is colored cyan in the P2C-1F11/RBD complex. P2C-1F11 and RBD-3M are colored red and blue, respectively, in the P2C-1F11/RBD-3M complex. The three RBD-3M mutated residues (N417, K484, and Y501) are shown as yellow spheres. (B) Interactions with P2C-1F11 around RBD-3M N417 and Y501 (left panel) and WT RBD K417 and N501 (right panel). (C) Interactions between K417 and the representative class I IGHV3-53/3-66 antibodies CB6, P5A-1D2, P22A-1D1, and CC12.1. (D) Interactions between E484 and the class II antibodies BD368-2, P5A-1B9, P2B-2F6, and CV07-270. In (C) and (D), antibodies are shown with different colors; hydrogen bond and salt bridges are represented by dashed and black lines, respectively. CB6/RBD (PDB: 7C01), P5A-1D2/RBD (PDB: 7CHO), P22A-1D1/RBD (PDB: 7CHS), CC12.1/RBD (PDB: 6XC2), BD368-2/RBD (PDB: 7CHC), P5A-1B9 (PDB: 7CZX), P2B-2F6 (PDB: 7BWJ), CV07-270 (PDB:6XKP). See also Figure S3 and Tables S1 and S2.

Figure 3

SARS-CoV-2 variants reduced sensitivity to convalescent plasma neutralization Reciprocal plasma dilutions (ID50) against SARS-CoV-2 variants are shown by (A) colored dots or (B) colored curves, each of which represents a different convalescent plasma. The geometric mean against each variant is indicated by a black horizontal line in (A) and black curve (B). Plasma samples from individuals with mild and severe disease are indicated by empty or solid circles in (A) and dashed or solid curves in (B). The fold change in ID50 between mutant and WT D614G pseudoviruses is shown by overall average at the top in (A) or individually in (C). “−” indicates an increase in resistance, and “+” indicates an increase in sensitivity. In (C), red highlights indicate a minimum 2-fold increase in resistance; blue a minimum 2-fold increase in sensitivity, and white a less than 2-fold change in resistance or sensitivity. BDL indicates that the highest concentration of plasma (1:60) failed to confer 50% neutralization. Standard plasma was obtained from the NIBSC (code: 20/136). Results of 23 plasma samples and standard plasma were calculated from three independent experiments performed in technical duplicates. ^∗p < 0.05; and ^∗∗∗∗p < 0.0001; ns, not significant. See also Figure S4.

Figure 4

Entry efficiency of SARS-CoV-2 variants into HeLa cells expressing ACE2 from diverse host species The values show the fold changes in luciferase activity for each indicated mutant pseudovirus variant compared with WT D614G. “+” indicates an increase in entry efficiency, and “−” indicates a decrease. Red highlights indicate at least a 3-fold increase in efficiency; blue indicates at least a 3-fold decrease in efficiency, and white indicates no change greater than 3-fold. Results were calculated from three independent experiments performed in technical triplicates.