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[Preprint]. 2021 Mar 8:2021.03.07.434227.
doi: 10.1101/2021.03.07.434227.

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies

Frauke Muecksch  1 , Yiska Weisblum  1 , Christopher O Barnes  2 , Fabian Schmidt  1 , Dennis Schaefer-Babajew  3 , Julio C C Lorenzi  3 , Andrew I Flyak  2 , Andrew T DeLaitsch  2 , Kathryn E Huey-Tubman  1   2   3   4   5 , Shurong Hou  4 , Celia A Schiffer  4 , Christian Gaebler  3 , Zijun Wang  3 , Justin Da Silva  1 , Daniel Poston  1 , Shlomo Finkin  3 , Alice Cho  3 , Melissa Cipolla  3 , Thiago Y Oliveira  3 , Katrina G Millard  3 , Victor Ramos  3 , Anna Gazumyan  3 , Magdalena Rutkowska  1 , Marina Caskey  3 , Michel C Nussenzweig  3   4 , Pamela J Bjorkman  2 , Theodora Hatziioannou  1 , Paul D Bieniasz  1   4
Affiliations

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies

Frauke Muecksch et al. bioRxiv. .

Update in

Abstract

Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled 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: Antibodies; Neutralization; SARS-CoV-2.

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Conflict of interest statement

Declaration of Interests The Rockefeller University has filed provisional patent applications in connection with this work on which M.C.N. (US patent 63/021,387) and Y.W., F.S., T.H. and P.D.B. (US patent 63/036,124) are listed as inventors.

Figures

Figure 1.
Figure 1.. Effects of somatic mutation of class 2 antibodies on potency and viral escape
(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 two 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 lineage 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) As in A for antibodies C548 and C549. (E) As in C for antibodies C548 and C549. Reduced antibody concentrations were required for C549 escape. (F,G) C548 (F) and C549 (G) neutralization of rVSV/SARS-CoV-2 1D7, 2E1 or plaque purified mutants thereof isolated following antibody selection, in 293T/ACE2cl.22 cells. Infected (%GFP+) cells relative to no antibody controls, mean and range of two independent experiments is plotted. See also Figure S1, S2, S3
Figure 2.
Figure 2.. Somatic mutation in a class 1 antibody lineage 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 two independent experiments. (B) RBD structure indicating positions of substitutions that affect sensitivity to neutralization by class 1 and C098 and C099 lineage 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, E, F) C098 (D) and C099 (E, F) neutralization of rVSV/SARS-CoV-2 1D7, 2E1 or plaque purified mutants thereof, isolated following antibody selection, in 293T/ACE2cl.22 cells. Infected (%GFP+) cells relative to no antibody controls, mean and range of two independent experiments is plotted. See also Figure S4
Figure 3.
Figure 3.. Effects of somatic mutation of class 3 antibodies on potency and 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 two independent experiments. (B) RBD structure indicating positions of substitutions that affect sensitivity to neutralization by class 3 and C132/C512 and C032/C080 lineage 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) C132 and C512 neutralization of rVSV/SARS-CoV-2 1D7, 2E1 or plaque purified mutants thereof, isolated following antibody selection, in 293T/ACE2cl.22 cells. Infected (%GFP+) cells relative to no antibody controls, mean and range of two independent experiments is plotted. (E) As in A for C032 and C080. (F) As in C for C032 and C080. (G) As in D for C032.
Figure 4.
Figure 4.. Effect of the E484K substitution alone or in combination with K417N/N501Y or L455R on matured class 1, 2 and 3 antibody sensitivity.
(A-F) 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) lineage antibodies in HT1080/ACE2cl.14 cells. Each of these variants was constructed in an R683G (furin cleavage site mutant) background to increase infectivity. Mean and range of two independent experiments.
Figure 5.
Figure 5.. Neutralization of heterologous sarbecoviruses by SARS-CoV-2 elicited antibodies and effects of somatic mutation on breadth.
(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) lineage antibodies in HT1080/ACE2cl.14 cells. Mean and range of two independent experiments.
Figure 6.
Figure 6.. Structures of class 1 and class 2 anti-RBD antibody 1.3m and 6.2m pairs
(A) Overlay of VH-VL domains of class 1 C098 and C099 Fabs bound to RBD from 2.0 Å 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,D) Interactions of C098 (panel C) and C099 (panel D) CDRH1 residues with RBD. Residues changed by somatic hypermutation indicated by an asterisk and enclosed in a red box. (E,F) Interactions of C098 (panel E) and C099 (panel 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 VH-VL domains of C051 are shown). (H) Overlay of VH-VL domains of C051 and C144 Fabs bound to S trimer. Both Fabs bridge between adjacent “down” RBDs, shown in inset as dark and light gray surfaces. (I,J) Interactions between RBD and C144 (panel I) and C051 (panel 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 Figure S5, S6
Figure 7
Figure 7. Structures of class 2 and class 3 anti-RBD 1.3m antibodies
(A) 3.4 Å cryo-EM density for class 2 C548-S complex (only the VH-VL 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 VH-VL 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 Figure S5, S7
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