Increased lung cell entry of B.1.617.2 and evasion of antibodies induced by infection and BNT162b2 vaccination

Abstract

The delta variant of SARS-CoV-2, B.1.617.2, emerged in India and has subsequently spread to over 80 countries. B.1.617.2 rapidly replaced B.1.1.7 as the dominant virus in the United Kingdom, resulting in a steep increase in new infections, and a similar development is expected for other countries. Effective countermeasures require information on susceptibility of B.1.617.2 to control by antibodies elicited by vaccines and used for COVID-19 therapy. We show, using pseudotyping, that B.1.617.2 evades control by antibodies induced upon infection and BNT162b2 vaccination, although with lower efficiency as compared to B.1.351. Further, we found that B.1.617.2 is resistant against Bamlanivimab, a monoclonal antibody with emergency use authorization for COVID-19 therapy. Finally, we show increased Calu-3-lung cell entry and enhanced cell-to-cell fusion of B.1.617.2, which may contribute to augmented transmissibility and pathogenicity of this variant. These results identify B.1.617.2 as an immune evasion variant with increased capacity to enter and fuse lung cells.

SciScore for 10.1101/2021.06.23.449568: (What is this?)

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

Table 1: Rigor

Ethics	IRB: Convalescent plasma was obtained from COVID-19 patients treated at the intensive care unit of the University Medicine Göttingen (UMG) under approval given by the ethic committee of the UMG (SeptImmun Study 25/4/19 Ü)
Sex as a biological variable	Cell culture: All cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO2. 293T (human, female, kidney; ACC-635, DSMZ, RRID: CVCL_0063), Vero76 cells (African green monkey kidney, female, kidney; CRL-1586, ATCC; RRID: CVCL_0574, kindly provided by Andrea Maisner) and BHK-21 (Syrian hamster, male, kidney; ATCC Cat# CCL-10; RRID: CVCL_1915, kindly provided by Georg Herrler) and A549-ACE2 cells (Hoffmann et al., 2021a), which were derived from parental A549 cells (human, male, lung; CRM-CCL-185, ATCC, RRID:CVCL_0023; kindly provided by Georg Herrler), were cultured in Dulbecco’s modified Eagle medium (PAN-Biotech) supplemented with 10% fetal bovine serum (FBS, Biochrom), 100 U/ml penicillin and 0.1 mg/ml streptomycin (pen/strep) (PAN-Biotech).
Randomization	For each sample, three randomly selected areas were imaged and S protein-driven syncytium formation was quantified by counting the total number of nuclei in syncytia per image.
Blinding	To eliminate potential bias and correct for counting errors, counting was performed blinded by two persons independently and for each sample average counts were used.
Power Analysis	not detected.
Cell Line Authentication	Authentication: Cell lines were validated by STR-typing, amplification and sequencing of a fragment of the cytochrome c oxidase gene, microscopic examination and/or according to their growth characteristics. Contamination: Furthermore, all cell lines were routinely tested for contamination by mycoplasma contamination

Table 2: Resources

Antibodies
Sentences	Resources
Subsequently, cells received culture medium containing anti-VSV-G antibody (culture supernatant from I1-hybridoma cells; ATCC no. CRL-2700; except for cells expressing VSV-G, which received only medium) in order to neutralize residual input virus.	anti-VSV-G suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cell culture: All cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO2. 293T (human, female, kidney; ACC-635, DSMZ, RRID: CVCL_0063), Vero76 cells (African green monkey kidney, female, kidney; CRL-1586, ATCC; RRID: CVCL_0574, kindly provided by Andrea Maisner) and BHK-21 (Syrian hamster, male, kidney; ATCC Cat# CCL-10; RRID: CVCL_1915, kindly provided by Georg Herrler) and A549-ACE2 cells (Hoffmann et al., 2021a), which were derived from parental A549 cells (human, male, lung; CRM-CCL-185, ATCC, RRID:CVCL_0023; kindly provided by Georg Herrler), were cultured in Dulbecco’s modified Eagle medium (PAN-Biotech) supplemented with 10% fetal bovine serum (FBS, Biochrom), 100 U/ml penicillin and 0.1 mg/ml streptomycin (pen/strep) (PAN-Biotech).	293T detected: (CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063) Vero76 suggested: None CVCL_0063) detected: (IZSLER Cat# BS CL 87, RRID:CVCL_0574) BHK-21 suggested: ATCC Cat# CCL-10, RRID:CVCL_1915) BHK-21 detected: (IZSLER Cat# BS CL 8, RRID:CVCL_1915) A549 suggested: None A549 detected: (BCRC Cat# 60074, RRID:CVCL_0023)
In addition, Calu-3 (human, male, lung; HTB-55, ATCC; RRID: CVCL_0609, kindly provided by Stephan Ludwig) and Caco-2 cells (human, male,	Calu-3 detected: (ATCC Cat# HTB-55, RRID:CVCL_0609) Caco-2 suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
colon; HTB-37, ATCC, RRID: CVCL_0025) were cultured in minimum essential medium (GIBCO) supplemented with 10% FBS, 1% pen/strep, 1x non-essential amino acid solution (from 100x stock, PAA) and 1 mM sodium pyruvate (Thermo Fisher Scientific)	ATCC detected: (RCB Cat# RCB0988, RRID:CVCL_0025)
Transfection of 293T cells was carried out by the calcium-phosphate precipitation method, while BHK-21 and A549-ACE2 cells were transfected using Lipofectamine LTX (Thermo Fisher Scientific).	293T suggested: None A549-ACE2 suggested: None
Following incubation, mixtures were inoculated onto Vero cells with particles incubated only with medium serving as control.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
Recombinant DNA
Sentences	Resources
Expression plasmids: Plasmids encoding pCAGGS-DsRed, pCAGGS-VSV-G (vesicular stomatitis virus glycoprotein), pCG1-WT SARS-CoV-2 S (codon optimized, based on the Wuhan/Hu-1/2019 isolate, equipped with D614G mutation; with C-terminal truncation of the last 18 amino acid), pCG1-SARS-CoV-2 S, B.1.351 (codon optimized; with C-terminal truncation of the last 18 amino acid), ACE2 (angiotensin converting enzyme 2) and soluble ACE2 have been previously described (Hoffmann et al., 2021a, Hoffmann et al., 2021b, Hoffmann et al., 2020).	pCAGGS-DsRed suggested: None pCAGGS-VSV-G suggested: None pCG1-WT suggested: None
The resulting open reading frame was further inserted into vector pCG1 plasmid (kindly provided by Roberto Cattaneo, Mayo Clinic College of Medicine, Rochester, MN, USA), using BamHI and XbaI restriction enzymes.	pCG1 suggested: None
Briefly, 293T cells were transfected with expression plasmids encoding S protein, VSV-G or empty plasmid (control).	VSV-G suggested: RRID:Addgene_138479)
At 24 h posttransfection, cells were inoculated with a replication-deficient vesicular stomatitis virus that lacks the genetic information for VSV-G and instead codes for two reporter proteins, enhanced green fluorescent protein and firefly luciferase (FLuc), VSV∗ΔG-FLuc (kindly provided by Gert Zimmer) at a multiplicity of infection of 3.	VSV∗ΔG-FLuc suggested: None
Software and Algorithms
Sentences	Resources
Protein models were generated employing the YASARA software (http://www.yasara.org/index.html) and are based on published crystal structure PDB: 6XDG (Hansen et al., 2020), PDB: 7L3N (Jones et al., 2020) or PDB: 7C01 (Shi et al., 2020), or a template that was constructed by modelling the SARS-2 S sequence on PDB: 6XR8 (Cai et al., 2020), using the SWISS-MODEL online tool (https://swissmodel.expasy.org	YASARA suggested: (YASARA, RRID:SCR_017591)
Data ware analyzed using Microsoft Excel (as part of the Microsoft Office software package, version 2019, Microsoft Corporation) and GraphPad Prism 8 version 8.4.3 (GraphPad Software).	Microsoft Excel suggested: (Microsoft Excel, RRID:SCR_016137) GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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No protocol registration statement was detected.

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

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Increased lung cell entry of B.1.617.2 and evasion of antibodies induced by infection and BNT162b2 vaccination

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