Affinity maturation of SARS-CoV-2 neutralizing antibodies confers potency, breadth, and resilience to viral escape mutations

Abstract

Antibodies elicited by infection accumulate somatic mutations in germinal centers that can increase affinity for cognate antigens. We analyzed 6 independent groups of clonally related severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) Spike receptor-binding domain (RBD)-specific antibodies from 5 individuals shortly after infection and later in convalescence to determine the impact of maturation over months. In addition to increased affinity and neutralization potency, antibody evolution changed the mutational pathways for the acquisition of viral resistance and restricted neutralization escape options. For some antibodies, maturation imposed a requirement for multiple substitutions to enable escape. For certain antibodies, affinity maturation enabled the neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.

Keywords: SARS-CoV-2; antibodies; neutralization.

Graphical abstract

Figure 1

Somatic mutation of class 2 antibodies affects potency and viral escape potential (A) Neutralization potency (IC₅₀) of C144, C051, and C052 measured using HIV-1-based SARS-CoV-2 variant pseudotypes and HT1080/ACE2cl.14 cells. The E484K substitution was constructed in an R683G (furin cleavage site mutant) background to increase infectivity. Mean of 2 independent experiments. (B) RBD structure indicating positions of substitutions that affect sensitivity to neutralization by class 2 and C144/C05/C052, C143/C164/C055, and C548/549 antibodies. (C) BLI affinity measurements for indicated antibodies against Q493R and E484K RBD, shown as continuous and dotted lines, respectively. (D) Decimal fraction (color gradient; white = 0, red = 1) of Illumina sequence reads encoding the indicated RBD substitutions following rVSV/SARS-CoV-2 replication (1D7 and 2E1 virus isolates) in the presence of the indicated amounts of antibodies for the indicated number of passages. (E) As in (A) for antibodies C548 and C549. (F) BLI affinity measurements for C548 (1.3 month) and C549 (6.2 month) for the indicated RBD proteins. (G) As in (D) for antibodies C548 and C549. Reduced antibody concentrations were required for C549 escape. (H and I) C548 (H) and C549 (I) neutralization of rVSV/SARS-CoV-2 1D7, 2E1, or plaque-purified mutants thereof isolated following antibody selection. Infected (%GFP⁺) cells relative to no antibody controls; mean and range of 2 independent experiments plotted. See also Figures S2 and S3.

Figure 2

Somatic mutation in a class 1 antibody confers potency and resilience to viral escape (A) Neutralization potency (IC₅₀) of C098 and C099 measured using HIV-1-based SARS-CoV-2 variant pseudotypes and HT1080/ACE2cl.14 cells. The E484K substitution was constructed in an R683G (furin cleavage site mutant) background to increase infectivity. Mean of 2 independent experiments. (B) RBD structure indicating positions of substitutions that affect sensitivity to neutralization by class 1 and C098 and C099 antibodies. (C) Decimal fraction (color gradient; white = 0, red = 1) of Illumina sequence reads encoding the indicated RBD substitutions following rVSV/SARS-CoV-2 replication (1D7 and 2E1 virus isolates) in the presence of the indicated amounts of antibodies for the indicated number of passages. (D–F) C098 (D) and C099 (E and F) neutralization of rVSV/SARS-CoV-2 1D7, 2E1 or plaque purified mutants thereof, isolated following antibody selection. Infected (%GFP⁺) cells relative to no antibody controls; mean and range of 2 independent experiments plotted. See also Figure S4.

Figure 3

Class 3 antibody maturation improves potency and reduces opportunities for viral escape (A) Neutralization potency (IC₅₀) of C132 and C512 measured using HIV-1-based SARS-CoV-2 variant pseudotypes and HT1080/ACE2cl.14 cells. The E484K substitution was constructed in an R683G (furin cleavage site mutant) background to increase infectivity. Mean of 2 independent experiments. (B) BLI affinity measurements for C132 (1.3 month) and C512 (6.2 month) for the indicated RBD proteins. (C) RBD structure indicating positions of substitutions that affect sensitivity to neutralization by class 3 and C132/C512 and C032/C080 antibodies. (D) Decimal fraction (color gradient; white = 0, red = 1) of Illumina sequence reads encoding the indicated RBD substitutions following rVSV/SARS-CoV-2 replication (1D7 and 2E1 virus isolates) in the presence of the indicated amounts of antibodies for the indicated number of passages. (E) C132 and C512 neutralization of rVSV/SARS-CoV-2 1D7, 2E1, or plaque-purified mutants thereof, isolated following antibody selection. Infected (%GFP⁺) cells relative to no antibody controls; mean and range of 2 independent experiments plotted. (F) As in (A) for C032 and C080. (G) BLI affinity measurements for C032 (1.3 month) and C080 (6.2 month) for indicated RBD proteins. (H) As in (D) for C032 and C080. (I) As in (E) for C032.

Figure 4

E484K, K417N, N501Y, and L455R substitutions have distinct effects on matured class 1, 2, and 3 antibody sensitivity Neutralization of HIV-1-based SARS-CoV-2 variant pseudotypes by C144/C051/C052 (A), C143/C164/C055 (B), C548/C549 (C), C098/C099 (D), C132/C512 (E), and C032/C080 (F) antibodies. Each of these variants was constructed in an R683G (furin cleavage site mutant) background to increase infectivity. Mean and standard deviation of 2 independent experiments.

Figure 5

Somatic mutation of SARS-CoV-2 elicited antibodies affects neutralization breadth against heterologous sarbecoviruses (A–F) Neutralization of HIV-1-based SARS-CoV, bat coronavirus (bCoV WIV16), or pangolin coronaviruses (pCov-GD and pCoV-GX) pseudotypes by C144/C051/C052 (A), C143/C164/C055 (B), C548/C549 (C), C098/C099 (D), C132/C512 (E), and C032/C080 (F) antibodies. Mean and standard deviation of 2 independent experiments. (G) Alignment of the heterologous sarbecovirus RBDs, with the positions and conservation of resistance mutations selected by the various clonally related antibody groups indicated by shading and hashmarks.

Figure 6

Structures of class 1 and class 2 anti-RBD antibody 1.3- and 6.2-month pairs reveal maturation-induced changes in antibody-Spike contacts (A) Overlay of V_H-V_L domains of class 1 C098 and C099 Fabs bound to RBD from 2.0 and 2.6 Å crystal structures, respectively. (B) CDR loops of C098 and C099 mapped onto the RBD surface. Fab epitopes are colored on the RBD surface. (C and D) Interactions of C098 (C) and C099 (D) CDRH1 residues with RBD. Residues changed by somatic hypermutation indicated by an asterisk and enclosed in a red box. (E and F) Interactions of C098 (E) and C099 (F) CDRH2 residues with RBD. Residues changed by somatic hypermutation indicated by an asterisk and enclosed in a red box. (G) 3.5 Å cryo-EM density for class 2 C051-S complex structure (only the V_H-V_L domains of C051 are shown). (H) Overlay of V_H-V_L domains of C051 and C144 Fabs bound to S trimer. Both Fabs bridge between adjacent “down” RBDs, shown in the inset as dark and light gray surfaces. (I and J) Interactions between RBD and C144 (I) and C051 (J) with a subset of interacting residues highlighted as sticks. Potential hydrogen bonds shown as dotted lines. Residues changed by somatic hypermutation indicated by an asterisk and enclosed in a red box. See also Figures S5 and S6.

Figure 7

Structures of class 2 and class 3 anti-RBD 1.3-month antibodies reveal structural basis for maturation-associated changes in activity (A) 3.4 Å cryo-EM density for class 2 C548-S complex (only the V_H-V_L domains of C548 are shown). (B) Close-up view of quaternary epitope involving bridging interactions between adjacent RBDs. (C) CDR loops mapped onto adjacent RBD surfaces. (D) Epitope of C548 highlighted on adjacent RBDs. (E) C548 paratope mapped onto adjacent RBDs. (F) Interactions between RBD and C548 with a subset of interacting residues highlighted as sticks. Potential hydrogen bonds shown as dotted lines. (G) 3.4 Å cryo-EM density for class 3 C032-S complex (only the V_H-V_L domains of C032 are shown). (H) Overlay of C032-RBD portion of the C032-S complex structure with an ACE2-RBD structure (from PDB: 6VW1). (I) Epitope of C032 highlighted on the RBD surface. (J) C032 paratope mapped onto RBD surface. (K) Interactions between RBD and C032 CDRH1 and CDRH3 loops, with a subset of interacting residues highlighted as sticks. Potential hydrogen bonds shown as dotted lines. See also Figures S5 and S7.