Delineating antibody escape from Omicron sublineages

Abstract

SARS-CoV-2 neutralizing antibodies play a critical role in prevention and treatment of COVID-19 but are challenged by viral evolution and antibody evasion, exemplified by the highly resistant Omicron BA.1 sublineage. ^1–12 Importantly, the recently identified Omicron sublineages BA.2.12.1 and BA.4/5 with differing spike mutations are rapidly emerging in various countries. By determining polyclonal serum activity of 50 convalescent or vaccinated individuals against BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.4/5, we reveal a further reduction of BA.4/5 susceptibility to vaccinee sera. Most notably, delineation of the sensitivity to an extended panel of 163 antibodies demonstrates pronounced antigenic differences of individual sublineages with distinct escape patterns and increased antibody resistance of BA.4/5 compared to the most prevalent BA.2 sublineage. These results suggest that the antigenic distance from BA.1 and the increased resistance compared to BA.2 may favor immune escape-mediated expansion of BA.4/5 after the first Omicron wave. Finally, while most monoclonal antibodies in clinical stages are inactive against all Omicron sublineages, we identify promising novel antibodies with high pan-Omicron neutralizing potency. Our study provides a detailed understanding of the antibody escape from the most recently emerging Omicron sublineages that can inform on effective strategies to prevent and treat COVID-19.

SciScore for 10.1101/2022.04.06.487257: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	IRB: sample collection: Serum samples from COVID-19-convalescent individuals were collected at the University Hospital Cologne under study protocols approved by the ethics committee (EC) of the Medical Faculty of the University of Cologne (16-054 and 20-1187). Field Sample Permit: Serum samples from vaccinated individuals were collected under protocols approved by the EC of Charité - Universitätsmedizin Berlin (EICOV, EA4/245/20) as well as the EC of the Federal State of Berlin and the Paul Ehrlich Institute (COVIM, EudraCT-No. 2021-001512-28). Consent: All study participants provided written informed consent.
Sex as a biological variable	not detected.
Randomization	neutralizing antibody panel and sequence analysis: The panel of 158 SARS-CoV-2-neutralizing monoclonal antibodies isolated from SARS-CoV-2 convalescent individuals included in the analysis in Figure 3 is based on 79 antibodies obtained in our previous work (Kreer et al., 2020; Vanshylla et al., 2022), 67 randomly selected (retrieved on January 1, 2021) human SARS-CoV-2-neutralizing antibodies deposited at CoV-AbDab (Raybould et al., 2021), and 12 antibodies in clinical use or development.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
All serum samples were tested for antibodies targeting the SARS-CoV-2 nucleocapsid using the SeraSpot Anti-SARS-CoV-2 IgG microarray-based immunoassay (Seramun Diagnostica).	Anti-SARS-CoV-2 IgG suggested: None
Samples from individuals with a history of SARS-CoV-2 infection, a positive SARS-CoV-2 nucleic acid amplification test (performed at sampling), or detectable anti-nucleocapsid antibodies were not included in this analysis.	anti-nucleocapsid suggested: None
For antibodies ADG-2, COV2-2130, COV2-2196, COV2-2381, MAD0004J08, and P2C-1F11, gene fragments based on the nucleotide sequences published in GenBank were ordered at IDT and cloned as above.	P2C-1F11 suggested: None
For antibodies C135, CT-P59, and LY-CoV1404, gene fragments based on antibody structures deposited in the Protein Data Bank (accession nos. 7K8Z, 7CM4, and 7MMO) were ordered at IDT after codon optimization using the IDT Codon Optimization Tool and cloned as above.	CT-P59 suggested: None LY-CoV1404 suggested: None 7CM4 suggested: None
For antibodies 47D11, BD-368-2, C144, and P2B-2F6, amino acid sequences were derived from CoV-AbDaB, corresponding nucleotide sequences generated and codon-optimized using the IDT Codon Optimization Tool, and gene fragments cloned as above.	C144 suggested: (Leinco Technologies Cat# C144, RRID:AB_2828501) P2B-2F6 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Monoclonal antibody production was performed using 293-6E cells (National Research Council of Canada) by co-transfection of heavy and light chain expression plasmids using 25 kDa branched polyethylenimine (Sigma-Aldrich)	293-6E suggested: RRID:CVCL_HF20)
Pseudovirus neutralization assays: Neutralization assays were performed using lentivirus-based pseudoviruses and ACE2-expressing 293T cells (Crawford et al., 2020; Vanshylla et al., 2021).	293T suggested: None
Pseudovirus particle production was performed in HEK293T cells by co-transfection of individual expression plasmids encoding for the SARS-CoV-2 spike protein, HIV-1 Tat, HIV-1 Gag/Pol, HIV-1 Rev, and luciferase-IRES-ZsGreen using FuGENE 6 Transfection Reagent (Promega)	HEK293T suggested: RRID:CVCL_HA71)
Pseudoviruses were titrated by infection of 293T-ACE2 cells and luciferase activity was determined after a 48-hour incubation at 37°C and 5% CO2 by addition of luciferin/lysis buffer (10 mM MgCl2, 0.3 mM ATP, 0.5 mM coenzyme A, 17 mM IGEPAL CA-630 (all Sigma-Aldrich), and 1 mM D-Luciferin (GoldBio) in Tris-HCL) using a microplate reader (Berthold)	293T-ACE2 suggested: None
Recombinant DNA
Sentences	Resources
Expression plasmids for Omicron sublinage spike proteins were produced by assembling and cloning codon-optimized overlapping gene fragments (Thermo Fisher) into the pCDNA3.1/V5-HisTOPO vector (Thermo Fisher) using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs), and included the full spike protein amino acid sequences with the following amino acid changes relative to Wu01: BA.1: A67V, Δ69-70, T95I, G142D, Δ143-145, N211I, Δ212, ins215EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F. BA.1.1: As for BA.1 with an additional R346K mutation.	pCDNA3.1/V5-HisTOPO suggested: None
Software and Algorithms
Sentences	Resources
Average background relative light units (RLUs) of non-infected cells were subtracted, and serum ID50s and antibody IC50s were determined as the serum dilutions and antibody concentrations resulting in a 50% RLU reduction compared to the average of virus-infected untreated controls cells using a non-linear fit model plotting an agonist vs. normalized dose response curve with variable slope using the least squares fitting method in Prism 7.0 (GraphPad).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
Phylogenetic analysis of antibodies belonging to the VH3-53/3-66\|VK1-9 public clonotype was performed by alignment of amino acid sequences with the MAFFT algorithm (Katoh et al., 2002) via the EMBL-EBI search and sequence analysis tools API (Madeira et al., 2019) and the Tree Builder tool from Geneious Prime 2020.0.4 (Biomatters) using the Jukes-Cantor distance model for tree building with the neighbour-joining method without resampling.	MAFFT suggested: (MAFFT, RRID:SCR_011811)
Data aggregation and visualization was performed with the Python libraries pandas (v1.1.5), NumPy (v1.19.2), SciPy (v1.5.2), Matplotlib (v3.3.4) with Python (v3.6.8), as well as Microsoft Excel 2011 for Mac (v14.7.3), and Adobe Illustrator.	Python suggested: (IPython, RRID:SCR_001658) NumPy suggested: (NumPy, RRID:SCR_008633) SciPy suggested: (SciPy, RRID:SCR_008058) Matplotlib suggested: (MatPlotLib, RRID:SCR_008624) Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137) Adobe Illustrator suggested: (Adobe Illustrator, RRID:SCR_010279)
Spearman’s rank correlation coefficients were determined using Prism 7.0 (GraphPad; serum samples) or the spearmanr() function of the SciPy-package (v 1.5.2)	Prism suggested: (PRISM, RRID:SCR_005375)

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Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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Results from scite Reference Check: We found no unreliable references.

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Delineating antibody escape from Omicron sublineages

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