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
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Abstract
Epidemiological data demonstrate that SARS-CoV-2 variants of concern (VOC) B.1.1.7 and B.1.617.2 are more transmissible and infections are associated with a higher mortality than non-VOC virus infections. Phenotypic properties underlying their enhanced spread in the human population remain unknown. B.1.1.7 virus isolates displayed inferior or equivalent spread in most cell lines and primary cells compared to an ancestral B.1 SARS-CoV-2, and were outcompeted by the latter. Lower infectivity and delayed entry kinetics of B.1.1.7 viruses were accompanied by inefficient proteolytic processing of spike. B.1.1.7 viruses failed to escape from neutralizing antibodies, but slightly dampened induction of innate immunity. The bronchial cell line NCI-H1299 supported 24- and 595-fold increased growth of B.1.1.7 and B.1.617.2 viruses, respectively, in the absence of detectable ACE2 expression and in a spike-determined fashion. Superior spread in NCI-H1299 cells suggests that VOCs employ a distinct set of cellular cofactors that may be unavailable in standard cell lines.
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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 … 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-1suggested: Noneanti-HAsuggested: (Sigma-Aldrich Cat# H3663, RRID:AB_262051)H3663suggested: Noneanti-tubulin ( Cell Signaling Technology , 2144Ssuggested: NoneBlocked 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-S2suggested: NoneNB100-56578suggested: (Novus Cat# NB100-56578, RRID:AB_838846)anti-SARS-CoV-2 nucleocapsid ( GeneTex , GTX135361suggested: NoneExperimental Models: Cell Lines Sentences Resources , NCI-H1299 (ATCC CRL-5803), NCI-H1299suggested: NCI-DTP Cat# NCI-H1299, RRID:CVCL_0060)HEK-293T (ATCC CRL-3216), HEK-293Tsuggested: 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-2suggested: NoneA549suggested: NoneAll samples were titrated on Vero E6 cells by plaque assay to determine infectious titers. Vero E6suggested: 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-3suggested: NoneCHO 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) CHOsuggested: NoneTZM-bl cells, stably expressing LTR-driven luciferase, were transfected with a plasmid encoding human ACE2 and human myc-TMPRSS2. TZM-blsuggested: RRID:CVCL_B478)Lentivirus production and transduction experiments: SARS-CoV-2 spike-HA-pseudotyped lentiviral particles were produced in triple-transfected HEK293T cells. HEK293Tsuggested: 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 + TMPRSS2suggested: NoneRecombinant 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. pCGsuggested: 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). pUC57suggested: 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-HAsuggested: NoneCells 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-HAsuggested: NonedeltaR8.91suggested: NonepCSII-luciferasesuggested: NoneSoftware 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). Geneioussuggested: (Geneious, RRID:SCR_010519)Quantification was done by the use of ImageJ 1.48v software. ImageJsuggested: (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...
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