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. 2021 Oct 5;37(1):109771.
doi: 10.1016/j.celrep.2021.109771. Epub 2021 Sep 28.

Paired heavy- and light-chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses

Bailey B Banach  1 , Gabriele Cerutti  2 , Ahmed S Fahad  3 , Chen-Hsiang Shen  4 , Matheus Oliveira De Souza  3 , Phinikoula S Katsamba  2 , Yaroslav Tsybovsky  5 , Pengfei Wang  6 , Manoj S Nair  6 , Yaoxing Huang  6 , Irene M Francino-Urdániz  7 , Paul J Steiner  7 , Matías Gutiérrez-González  3 , Lihong Liu  6 , Sheila N López Acevedo  3 , Alexandra F Nazzari  4 , Jacy R Wolfe  3 , Yang Luo  6 , Adam S Olia  4 , I-Ting Teng  4 , Jian Yu  8 , Tongqing Zhou  4 , Eswar R Reddem  2 , Jude Bimela  2 , Xiaoli Pan  3 , Bharat Madan  3 , Amy D Laflin  3 , Rajani Nimrania  3 , Kwok-Yung Yuen  9 , Timothy A Whitehead  7 , David D Ho  6 , Peter D Kwong  10 , Lawrence Shapiro  11 , Brandon J DeKosky  12
Affiliations

Paired heavy- and light-chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses

Bailey B Banach et al. Cell Rep. .

Abstract

Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2.

Keywords: B-cell; SARS-CoV-2; biotechnology; immunity; neutralization; public antibody; virology; yeast display.

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Conflict of interest statement

Declaration of interests B.B.B., A.S.F., M.O., P.W., L.L., S.N.L.A., J.R.W., X.P., B.M., D.D.H., and B.J.D. declare competing financial interests in the form of a provisional patent application filed by the University of Kansas.

Figures

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Graphical abstract
Figure 1
Figure 1
A novel SARS-CoV-2 neutralizer in the reproducible IGHV3-53/3-66 antibody class targets the ACE2 binding site of both ordered and disassembled spike (A) Antibody 910-30 shows moderately potent neutralization capacity compared in both VSV-pseudo-type virus and authentic virus assays. Data are represented as means ± SDs. (B) Representative micrograph of pH 5.5 negative-staining electron microscopy showing 910-30 Fab bound to SARS-CoV-2 S2P protein. Inset shows representative 2D class averages; arrows point to bound Fab fragments. Scale bars: 50 nm (micrographs) 20 nm (2D class averages). (C) Cryo-EM map and molecular model of 910-30 Fab in complex with SARS-CoV-2 spike at 4.75 Å resolution. (D) Cryo-EM map obtained of 9:1 molar ratio 910-30 Fab:spike at pH 5.5. (E) The structural superposition of ACE2 (PDB: 6M0J) and 910-30 (PDB: 7KS9) in complex with SARS-CoV-2 RBD shows a representative ACE2 competition mechanism defining IGHV3-53/3-66 class neutralization. See also Figures S1 and S2 and Table S1.
Figure 2
Figure 2
IGHV3-53/3-66 class neutralization potency is driven by strong competition with ACE2 for spike S2P recognition (A) Comparison between ACE2 binding site and the IGHV3-53/3-66 class epitope on RBD show substantial overlap. (B) Dot chart of reported wild-type authentic virus neutralization IC50 titers for previously published IGHV3-53/3-66 anti-RBD antibodies. Data were plotted without correcting for any differences in neutralization assay protocols. The line indicates the mean of IC50 values. A list of antibodies, IC50 values, and citations are provided in Table S2. (C) IgG ELISA binding titrations for select IGHV3-53/3-66 class members against S2P spike and RBD antigens, with an IGHV gene-matched control (mAb 4-3) and an isotype control. Data are represented as means ± SDs. (D) Pseudovirus and authentic virus neutralization show that IC50 neutralization potency ranges 2 orders of magnitude between the selected IGHV3-53/3-66 class members, along with an IGHV gene-matched control (mAb 4-3). Data are represented as means ± SDs. (E) dhACE2 competition ELISA against SARS-CoV-2 S2P spike showing IgG binding response to increasing dhACE2 (ACE2) concentrations. dhACE2 concentration is provided as both μg/mL and as ACE2 molar excess units. Data are represented as means ± SDs. (F) Sequence alignment of heavy chain (upper) and light chain (lower) genes selected for detailed investigation. See also Figure S3 and Tables S2 and S3.
Figure 3
Figure 3
Heavy- and light-chain analyses reveal critical contributions of both VH and VL for potent antibody neutralization in the IGHV3-53/3-66 antibody class (A) SARS-CoV-2 pseudovirus neutralization results for heavy-light swapped IgG panel. Data are represented as means ± SDs. (B) dhACE2 competition ELISA against SARS-CoV-2 S2P protein showing heavy-light swapped IgG binding response to increasing dhACE2 (ACE2) concentrations. dhACE2 concentration is provided in μg/mL and also as ACE2 molar excess units. Data are represented as means ± SDs. (C) Conserved IGHV3-53/3-66 class light-chain kappa (left panel) and lambda (right panel) genetic elements associated with contacting the RBD interface. CDR-L1 residues are not specific to IGKV1-33 (910-30) and IGLV2-8 (C105) (Table S4). (D) Left panel: structural superposition of IGHV3-53/3-66 Fab variable domains in complex with RBD shows the same binding orientation for 8 different class antibodies aligned on RBD. Right panel: close-up views of the Fab:RBD interface for the 8 IGHV3-53/3-66 antibodies superimposed on RBD. Conserved interactions of CDR-H1, -H2, and -L1 define the structural signatures responsible for viral neutralization by the IGHV3-53/3-66 antibody class. (E) Estimated probability of IGHV3-53/3-66 class pre-cursor antibodies derived from healthy donor (HIP1, HIP2, HIP3) immune repertories. See also Figure S3 and Table S4.
Figure 4
Figure 4
Up/down conformational changes of RBD influence IGHV3-53/3-56 antibody class recognition of spike protein across the serological to endosomal pH range (A) Schematic of RBD conformational states inferred by cryo-EM and experimental analysis for un-ligated D614 and D614G spike. U and D denote up and down RBD configurations, respectively. Percentages denote observed particle populations. (B) dhACE2 competition ELISA at endosomal pH against SARS-CoV-2 S2P D614 protein. dhACE2 concentration is provided as both μg/mL and ACE2 molar excess units. Data are represented as means ± SDs. (C) Single-cycle SPR kinetic assays for 1-20 and B38 IgG at serological and endosomal pH against biotinylated spike. Black traces represent experimental data and red traces represent the fit to a 1:1 interaction model. The number in parentheses represents the error of the fit in the last digit. (D) Correlations between authentic virus neutralization potency (Figure 2D) versus the ratio of antibody IgG affinity to spike S2P (Figure S4A) divided by dhACE2 affinity to spike S2P (reported from Zhou et al., 2020b). (E) Qualitative octet pH series for wild-type S2P spike and escape mutant D614G S2P spike across a range of pH values. See also Figures S3 and S4.
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