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. 2021 Jul 13;36(2):109385.
doi: 10.1016/j.celrep.2021.109385. Epub 2021 Jun 25.

Resistance of SARS-CoV-2 variants to neutralization by antibodies induced in convalescent patients with COVID-19

Yu Kaku  1 , Takeo Kuwata  2 , Hasan Md Zahid  1 , Takao Hashiguchi  3 , Takeshi Noda  4 , Noriko Kuramoto  1 , Shashwata Biswas  1 , Kaho Matsumoto  1 , Mikiko Shimizu  1 , Yoko Kawanami  1 , Kazuya Shimura  5 , Chiho Onishi  5 , Yukiko Muramoto  6 , Tateki Suzuki  7 , Jiei Sasaki  7 , Yoji Nagasaki  8 , Rumi Minami  9 , Chihiro Motozono  10 , Mako Toyoda  10 , Hiroshi Takahashi  11 , Hiroto Kishi  11 , Kazuhiko Fujii  11 , Tsuneyuki Tatsuke  12 , Terumasa Ikeda  13 , Yosuke Maeda  14 , Takamasa Ueno  10 , Yoshio Koyanagi  5 , Hajime Iwagoe  15 , Shuzo Matsushita  16
Affiliations

Resistance of SARS-CoV-2 variants to neutralization by antibodies induced in convalescent patients with COVID-19

Yu Kaku et al. Cell Rep. .

Abstract

Administration of convalescent plasma or neutralizing monoclonal antibodies (mAbs) is a potent therapeutic option for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, SARS-CoV-2 variants with mutations in the spike protein have emerged in many countries. To evaluate the efficacy of neutralizing antibodies induced in convalescent patients against emerging variants, we isolate anti-spike mAbs from two convalescent COVID-19 patients infected with prototypic SARS-CoV-2 by single-cell sorting of immunoglobulin-G-positive (IgG+) memory B cells. Anti-spike antibody induction is robust in these patients, and five mAbs have potent neutralizing activities. The efficacy of most neutralizing mAbs and convalescent plasma samples is maintained against B.1.1.7 and mink cluster 5 variants but is significantly decreased against variants B.1.351 from South Africa and P.1 from Brazil. However, mAbs with a high affinity for the receptor-binding domain remain effective against these neutralization-resistant variants. Rapid spread of these variants significantly impacts antibody-based therapies and vaccine strategies against SARS-CoV-2.

Keywords: COVID-19; SARS-CoV-2; mAb; neutralizing antibody; variant.

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Conflict of interest statement

Declaration of interests Y. Kaku, T.K., and S.M. are listed as inventors on a patent application related to this work. The remaining authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Isolation of neutralizing mAbs from two convalescent patients (A) Binding activity of plasma samples from patients A and B to the SARS-CoV-2 S protein was analyzed by flow cytometry. Binding of IgG to cells expressing SARS-CoV-2 S at ×10,000 dilution of plasma samples is shown as a histogram. Dotted line, no plasma control; orange line, plasma from a healthy donor. (B) Neutralization activity of plasma samples analyzed using pseudoviruses expressing the S protein from SARS-CoV-2, SARS-CoV, and MERS-CoV. IC50 values (dilution) are summarized. (C) Strategy to isolate neutralizing mAbs is shown schematically. (D) A representative flow cytometry plot is shown. CD3−CD14−CD8− cells were used to sort memory B cells (7AAD−CD19+IgM−IgG+CD27+ cells), as shown in dotplots. (E) Numbers of recombinant IgGs, S binders, RBD binders, and neutralizers from patients A and B are summarized.
Figure 2
Figure 2
Characterization of five neutralizing mAbs (A) Neutralization of SARS-CoV-2 pseudovirus by mAbs 6-74, 3-5, 8-92, 10-121, and 9-105 in 293FT/ACE2/TMPRSS2, Calu-3 (human lung cancer cell line), and Caco-2 (human colon adenocarcinoma cell line) cells is shown. A non-neutralizing mAb, 8-38, was used as a negative control (orange open circle). (B) Cell fusion of 293FT/DSP8-11/SARS-CoV-2-S cells with 293FT/DSP1-7/ACE2/TMPRSS2 and Calu-3/DSP1-7 cells was measured by luciferase activity at 6 and 20 h after coculture, respectively. A non-neutralizing mAb, 5-76, was used as a negative control (orange open circle). (C) IC50 values of the five mAbs are summarized. (D) The binding and genetic characteristics of mAbs are summarized. Binding to RBD and NTD was analyzed by AlphaScreen. KD values were determined by SPR analysis (Figure S1D). Gene usage (gene), somatic hypermutation % (SHM) of heavy and light chains, and CDRH3 amino acids (aa) and length were analyzed by IMGT vquest. Data shown in (A) and (B) are represented as means ± SD (n = 3). See also Figure S1.
Figure 3
Figure 3
Neutralizing activity against SARS-CoV-2 variants (A) Amino acid substitutions in the S protein of SARS-CoV-2 variants B.1.1.7, B.1.351, P.1, and mink cluster 5 are schematically shown. (B) IC50 values of mAbs (μg/mL) and plasma samples (dilution) against pseudoviruses with SARS-CoV-2 S variants are summarized with fold change (variant IC50 value/614G IC50 value). (C) Neutralization of pseudoviruses with SARS-CoV-2 S variants (upper panels) and RBD mutants (lower panels) was examined by mAbs in 293FT/ACE2/TMPRSS2 cells. Data are represented as means ± SD (n = 3). (D) IC50 values of plasma samples from 14 patients with COVID-19 other than patients A and B were compared between the 614G pseudovirus and pseudoviruses carrying the variant S protein. Statistical analysis was performed using a Wilcoxon matched-pairs signed rank test, and p values are shown.
Figure 4
Figure 4
Neutralization of authentic SARS-CoV-2 (A) A plaque assay was performed using the authentic SARS-CoV-2 Japan/NGS-IA-1/2020 strain and Vero cells. Representative result of plaque formation in the presence of mAb 9-105 is shown. (B) Neutralization of authentic SARS-CoV-2 by mAbs and plasma samples are shown. (C) IC50 values of mAbs and plasma samples are summarized. (D) Neutralization of authentic SARS-CoV-2 variants B.1.1.7, B.1.351, and P.1 by mAbs 10-121 and 9-105 are shown. (E) IC50 values of mAbs (μg/mL) against authentic SARS-CoV-2 variants are summarized with fold change (variant IC50 value/prototype IC50 value). Data shown in (B) and (D) are represented as means ± SD (n = 3). See also Figure S2 for structure of 9-105 and S complex.
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