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SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma

Andreano, Emanuele, Piccini, Giulia, Licastro, Danilo, Casalino, Lorenzo, Johnson, Nicole V., Paciello, Ida, Monego, Simeone Dal, Pantano, Elisa, Manganaro, Noemi, Manenti, Alessandro, Manna, Rachele, Casa, Elisa, Hyseni, Inesa, Benincasa, Linda, Montomoli, Emanuele, Amaro, Rommie E., McLellan, Jason S., Rappuoli, Rino·2020
DOI 10.1101/2020.12.28.424451PMC PMC7781313

Preprint — published as 10.1073/pnas.2103154118

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Abstract

To investigate the evolution of SARS-CoV-2 in the immune population, we co-incubated authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for 7 passages, but after 45 days, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed at day 80 by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom and South Africa of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed. One Sentence Summary: Three mutations allowed SARS-CoV-2 to evade the polyclonal antibody response of a highly neutralizing COVID-19 convalescent plasma.

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Crossref · 10.1101/2020.12.28.424451

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Figures

Fig. 1. Evolution of an authentic SARS-CoV-2 escape mutant. (A) Schematic representation of the 24-well plate format used to select the authentic SARS-CoV-2 escape mutant. Blue, red, green and yellow wells show feeder cells protect from PT188 neutralization, CPE, authentic virus on Vero E6 cells and Vero E6 alone, respectively. (B) The graph shows the PT188 neutralization titer after each mutation acquired by the authentic virus. Specific mutations, fold decrease and days to which the mutations occur are reported in the figure. (C) SARS-CoV-2 S-protein gene showing type, position of mutations and frequency of mutations.
Fig. 1. Evolution of an authentic SARS-CoV-2 escape mutant. (A) Schematic representation of the 24-well plate format used to select the authentic SARS-CoV-2 escape mutant. Blue, red, green and yellow wells show feeder cells protect from PT188 neutralization, CPE, authentic virus on Vero E6 cells and Vero E6 alone, respectively. (B) The graph shows the PT188 neutralization titer after each mutation acquired by the authentic virus. Specific mutations, fold decrease and days to which the mutations occur are reported in the figure. (C) SARS-CoV-2 S-protein gene showing type, position of mutations and frequency of mutations.CC BY-NC-ND · PubMed Central
Fig. 2. Neutralization efficacy of plasma and thirteen mAbs to SARS-CoV-2 PT188-EM. (A) Heat-map showing the neutralization activity of tested plasma samples to the SARS-CoV-2 WT, D614G and PT188-EM variants. (B) Heat-maps showing neutralization profiles of tested mAbs. (C) Negative stain EM 2D class averages showing J13, I21 and H20 Fabs bound to the SARS-CoV-2 S-protein. (D) 3D reconstruction of J13 bound to the NTD domain of the S-protein viewed looking along (left panel) or toward (right panel) the viral membrane.
Fig. 2. Neutralization efficacy of plasma and thirteen mAbs to SARS-CoV-2 PT188-EM. (A) Heat-map showing the neutralization activity of tested plasma samples to the SARS-CoV-2 WT, D614G and PT188-EM variants. (B) Heat-maps showing neutralization profiles of tested mAbs. (C) Negative stain EM 2D class averages showing J13, I21 and H20 Fabs bound to the SARS-CoV-2 S-protein. (D) 3D reconstruction of J13 bound to the NTD domain of the S-protein viewed looking along (left panel) or toward (right panel) the viral membrane.CC BY-NC-ND · PubMed Central
Fig. 3. In-silico modeling of the PT188-EM spike NTD and RBD. (A) In-silico model of the NTD of the SARS-CoV-2 PT188-EM spike protein based on PDB id 7JJI. This model accounts for the 11-amino-acid insertion (yellow ribbon) and F140 deletion (highlighted with a yellow bead). N5 loop as in the wild type cryo-EM structure (PDB id: 7JJI) is shown as a transparent red ribbon. (B) Close-up of the PT188-EM spike NTD model in complex with antibody 4A8. Both heavy chain (HC, light gray) and light chain (LC, dark gray) of 4A8 are shown. The 11-amino-acid insertion (yellow ribbon) within N5 loop introduces a new N-linked glycan (N248d) that sterically clashes with 4A8, therefore disrupting the binding interface. The N-glycan at position N149 is however compatible with 4A8 binding. (C) Conformational dynamics of the PT188-EM spike NTD model resulting from 100 ns of molecular dynamics simulation is shown by overlaying multiple frames along the generated trajectory. (D) In-silico model of the PT188-EM spike RBD based on PDB id 6M17, where the E484K mutation is shown with licorice representation.
Fig. 3. In-silico modeling of the PT188-EM spike NTD and RBD. (A) In-silico model of the NTD of the SARS-CoV-2 PT188-EM spike protein based on PDB id 7JJI. This model accounts for the 11-amino-acid insertion (yellow ribbon) and F140 deletion (highlighted with a yellow bead). N5 loop as in the wild type cryo-EM structure (PDB id: 7JJI) is shown as a transparent red ribbon. (B) Close-up of the PT188-EM spike NTD model in complex with antibody 4A8. Both heavy chain (HC, light gray) and light chain (LC, dark gray) of 4A8 are shown. The 11-amino-acid insertion (yellow ribbon) within N5 loop introduces a new N-linked glycan (N248d) that sterically clashes with 4A8, therefore disrupting the binding interface. The N-glycan at position N149 is however compatible with 4A8 binding. (C) Conformational dynamics of the PT188-EM spike NTD model resulting from 100 ns of molecular dynamics simulation is shown by overlaying multiple frames along the generated trajectory. (D) In-silico model of the PT188-EM spike RBD based on PDB id 6M17, where the E484K mutation is shown with licorice representation.CC BY-NC-ND · PubMed Central

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Research works printed but not linked: 2

  1. A. Manenti et al., Evaluation of SARS-CoV-2 neutralizing antibodies using a CPE-based colorimetric live virus micro-neutralization assay in human serum samples. Journal of medical virology, (2020). · DOI
  2. X. Chi et al., A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. 369, 650–655 (2020). · DOI · Paper page

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Metadata

Authors
Andreano, E., Piccini, G., Licastro, D., Casalino, L., Johnson, N. V., Paciello, I., Monego, S. D., Pantano, E., Manganaro, N., Manenti, A., Manna, R., Casa, E., Hyseni, I., Benincasa, L., Montomoli, E., Amaro, R. E., McLellan, J. S., & Rappuoli, R.
ORCID
Publisher
openRxiv
Published
2020-12-28
DOI
10.1101/2020.12.28.424451
PMC
PMC7781313
References
56
Type
Preprint

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