Post-entry, spike-dependent replication advantage of B.1.1.7 and B.1.617.2 over B.1 SARS-CoV-2 in an ACE2-deficient human lung cell line

Oct 23, 2021

SciScore for 10.1101/2021.10.20.465121: (What is this?)

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

Table 1: Rigor

Ethics	IRB: The use of clinical samples (sera) was approved by the Institutional Review Board at Charité -Universitätsmedizin Berlin (EA1/068/20, EA2/092/20 and EA2/066/20) and is in accordance with the Berlin State Hospital Law, allowing for pseudonymized scientific analysis of routine patient data. Consent: All other experiments were conducted with hBAECs isolated from explanted lungs which were obtained from the Hannover Lung Transplant Program after patients informed consent, ethical vote 2923-2015.
Sex as a biological variable	Per group, nine male and female Roborovski dwarf hamsters (Phodopus roborovskii) obtained via the German pet trade were used.
Randomization	Hamsters of the same sex were randomly distributed into experimental groups and individually marked with a subcutaneously implanted IPTT-300 transponder (BMDS, Seaford (DE), USA) that allows remote identification and measurement of body temperature.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Blocked membranes were incubated with the following antibodies: mouse anti-HIV-1 p24 capsid (ExBio, 1:1000), rabbit anti-S2 spike (Novusbio, NB100-56578, 1:1000), mouse anti-HA (Sigma, H3663, 1:1400), rabbit anti-tubulin (Cell Signaling Technology, 2144S, 1:1000).	anti-HIV-1 suggested: None anti-HA suggested: (Sigma-Aldrich Cat# H3663, RRID:AB_262051) H3663 suggested: None anti-tubulin ( Cell Signaling Technology , 2144S suggested: None
Blocked membranes were incubated with the following antibodies: rabbit anti-S2 spike (Novusbio, NB100-56578, 1:1000), rabbit anti-SARS-CoV-2 nucleocapsid (GeneTex, GTX135361, 1:1000).	anti-S2 suggested: None NB100-56578 suggested: (Novus Cat# NB100-56578, RRID:AB_838846) anti-SARS-CoV-2 nucleocapsid ( GeneTex , GTX135361 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
, NCI-H1299 (ATCC CRL-5803),	NCI-H1299 suggested: NCI-DTP Cat# NCI-H1299, RRID:CVCL_0060)
HEK-293T (ATCC CRL-3216),	HEK-293T suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)
Virus infection and virus growth kinetics in cell cultures: Vero E6, Caco-2, NCI-H1299 and A549 cells were seeded at a densitiy of 350,000 cells per ml and Calu-3 cells at a density of 500,000 cells per ml one day prior to infection.	Caco-2 suggested: None A549 suggested: None
All samples were titrated on Vero E6 cells by plaque assay to determine infectious titers.	Vero E6 suggested: RRID:CVCL_XD71)
Competition assay: Calu-3 cells were infected in 24-well plates with a mixture of two SARS-CoV-2 variants, using three different ratios (1:1 and 9:1 and 1:9) and an initial, total infectious dose of 10.000 PFU (corresponding to an MOI of 0.04).	Calu-3 suggested: None
CHO cells were transiently transfected with expression plasmids for HIV-1 Tat and individual pCG-spike-HA or empty vector control for 48 hours, using Lipofectamine LTX Reagent with PLUS™ Reagent (Invitrogen)	CHO suggested: None
TZM-bl cells, stably expressing LTR-driven luciferase, were transfected with a plasmid encoding human ACE2 and human myc-TMPRSS2.	TZM-bl suggested: RRID:CVCL_B478)
Lentivirus production and transduction experiments: SARS-CoV-2 spike-HA-pseudotyped lentiviral particles were produced in triple-transfected HEK293T cells.	HEK293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
Experimental Models: Organisms/Strains
Sentences	Resources
Cells and culture conditions: A549 parental (ATCC CCL-185), A549-ACE2, A549-TMPRSS2, A549-ACE2 + TMPRSS2 (Widera et al. 2021), Caco-2 (ATCC HTB-37), Calu-3 (HTB-55), CHO (HIV Reagent Program ARP-2238)	A549-TMPRSS2, A549-ACE2 + TMPRSS2 suggested: None
Recombinant DNA
Sentences	Resources
Codon-optimized, C-terminally tagged spike cDNAs in pCG were generated using pCG-SARS-CoV-2 spike Wuhan as a template (Hoffmann et al., 2020) in which the N-terminus was repaired and D614G was introduced via site-directed mutagenesis.	pCG suggested: RRID:Addgene_51476)
For generation of the D614G mutant we performed site-directed mutagenesis PCR (NEB) on synthetic viral subgenomic fragments cloned into pUC57 vectors (Thi Nhu Thao et al., 2020).	pUC57 suggested: RRID:Addgene_40306)
CHO cells were transiently transfected with expression plasmids for HIV-1 Tat and individual pCG-spike-HA or empty vector control for 48 hours, using Lipofectamine LTX Reagent with PLUS™ Reagent (Invitrogen)	pCG-spike-HA suggested: None
Cells were transfected with individual pCG-SARS-CoV-2 spike-HA plasmids, the HIV-1-based packaging plasmid deltaR8.91 (Zufferey et al., 1997) and the luciferase transfer plasmid pCSII-luciferase (Agarwal et al., 2006) via calcium phosphate precipitation.	pCG-SARS-CoV-2 spike-HA suggested: None deltaR8.91 suggested: None pCSII-luciferase suggested: None
Software and Algorithms
Sentences	Resources
The processed sequence reads were mapped to the BetaCoV/Munich/ChVir984/2020 genome (here referred to as SARS-CoV-2 2019-nCoV strain) in Geneious (version 9.1.8).	Geneious suggested: (Geneious, RRID:SCR_010519)
Quantification was done by the use of ImageJ 1.48v software.	ImageJ suggested: (ImageJ, RRID:SCR_003070)

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: We detected the following sentences addressing limitations in the study:

While caveats and limitations of neoplastic cell lines apply, NCI-H1299 cells yielded higher levels of replication and infectious virus production not only for B.1.1.7, but also for B.1.617.2. This cell line of epithelial morphology is devoid of detectable ACE2 protein and remains susceptible to SARS-CoV-2 infection even in the presence of ACE2-neutralizing antibodies, suggesting the existence of an alternative, but not variant-specific mode of entry. This finding is reminiscent of a recent study (Puray-Chavez et al., 2021) that identified SARS-CoV-2 infection of the H522 lung cell line in an ACE2-independent fashion (in this study the alternative way of infection could only be utilized by viruses carrying an E484D substitution within the spike protein RBD). Surprisingly, while experiments with reciprocal chimeras established that the B.1.1.7 spread advantage in NCI-H1299 cells is mediated to a large extent by its spike, the entry process per se was similarly or even less efficient for B.1.1.7 than for B.1. Notably, a virus consisting of the B.1.1.7 backbone with B.1 spike displayed a slightly better replication efficiency than the original B.1 virus in type-I IFN-competent Calu-3 cells, confirming that other genetic determinants of B.1.1.7 beyond spike contribute to enhanced replication. These findings suggest a post-entry, yet largely spike-dependent replication advantage of B.1.1.7 that manifests itself in NCI-H1299 cells. Remarkably, we conducted preliminary studies of re...

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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No protocol registration statement was detected.

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

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