key: cord-0297945-8d0mgjbz
authors: Niemeyer, Daniela; Schroeder, Simon; Friedmann, Kirstin; Weege, Friderike; Trimpert, Jakob; Richter, Anja; Stenzel, Saskia; Jansen, Jenny; Emanuel, Jackson; Kazmierski, Julia; Pott, Fabian; Jeworowski, Lara M.; Olmer, Ruth; Jaboreck, Mark-Christian; Tenner, Beate; Papies, Jan; Heinze, Julian; Walper, Felix; Schmidt, Marie L.; Heinemann, Nicolas; Möncke-Buchner, Elisabeth; Veith, Talitha; Baumgardt, Morris; Hoffmann, Karen; Widera, Marek; Thao, Tran Thi Nhu; Balázs, Anita; Schulze, Jessica; Mache, Christin; Morkel, Markus; Ciesek, Sandra; Hanitsch, Leif G.; Mall, Marcus A.; Hocke, Andreas C.; Thiel, Volker; Osterrieder, Klaus; Wolff, Thorsten; Martin, Ulrich; Corman, Victor M.; Müller, Marcel A.; Goffinet, Christine; Drosten, Christian
title: 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
date: 2021-10-20
journal: bioRxiv
DOI: 10.1101/2021.10.20.465121
sha: 36e79a545fbc7eb39dcac7ad44de574261612478
doc_id: 297945
cord_uid: 8d0mgjbz

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.

Since its emergence, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has 76 genetically diversified, giving rise to variants with altered phenotypic properties (Rambaut et al. 77 2020). In May 2021 the WHO announced a scheme for labeling SARS-CoV-2 lineages with 78 was particularly delayed in the very early phase of replication (Fig. 1F) . Cultivation of infected cells 130 at 32°C in order to resemble the temperature in the upper respiratory tract did not alter relative 131 replication efficiencies (Fig. 1G ). Under competitive passaging in Calu-3 cells, B.1 outcompeted 132 B.1.1.7, even when the starting inoculum contained a nine-fold excess of B.1.1.7 (Fig. 1H ). In sum, 133 immortalized cell models failed to establish a clear growth advantage of B.1.1.7 that correlates 134 with its enhanced transmissibility and pathogenicity in vivo. quantitative immunoblots (Fig. 3A, Fig. S1A ). However, quantification of the proportion of S2-HA 155 spike relative to the total spike-HA signal revealed a 1.8-fold reduction of proteolytic processing of 156 the B.1.1.7 glycoprotein compared to that of B.1 (Fig. 3A) . No single B.1.1.7-defining mutation, 157 including P 681 H, fully recapitulated this property, even though deletion of H 69 /V 70 showed a trend 158 towards more efficient processing, in agreement with , and T 716 H showed a 159 trend towards decreased proteolytic processing. The data suggest that a combination of amino 160 acid exchanges is required for rendering proteolytic processing of B.1.1.7 spike less efficient. 161

Reduced processing of B.1.1.7 spike was accompanied by a 2.3-fold decrease of spike levels 162 associated with lentiviral particles, when compared to particles containing B.1 spike (Fig. 3B) . 163

Interestingly, the individual T 716 H mutation was sufficient for reduction of spike quantities 164 associating to lentiviral particles. In accordance with previous reports (Kemp et We next hypothesized that B.1.1.7 has evolved superior ability to prevent or evade cell-219 intrinsic immunity. In the basal medium of infected differentiated bronchial airway epithelial cell 220 cultures, no significant variant-specific differences were identified for various cytokines and other 221 secreted proteins related to innate immunity, including IFN-α, IFN-γ, and IP-10 (Fig. 5C ). In 222 infected Calu-3 cells, expression of IFNB, MXA, CCL5, IL6 and TNFA was induced to similar levels 223 by both variants (Fig. 5D ). Interestingly, B.1.1.7 infection appeared to induce slightly lower levels 224 of IFNL expression than B.1 (Fig. 5D) Interestingly, there was no advantage in entry of lentiviral particles based on the B.1.1.7 239 spike protein in NCI-H1299 cells (Fig. 6B) . Also, there was no advantage for authentic SARS-240

CoV-2 B.1.1.7. virus in entering NCI-H1299 cells in a synchronized entry assay (Fig. 6C) , 241

indicating that the more efficient replication of B.1.1.7 may not necessarily be determined by 242 improved entry. Even more surprisingly, replication of B.1.1.7 in NCI-H1299 cells occurred in the 243 absence of detectable ACE2 protein (Fig. 6D) , and ACE2 and TMPRSS2 mRNAs were only 244 weakly expressed as compared to other SARS-CoV-2-susceptible cell cultures (Fig. 6E ). In order 245 to exclude the possibility that SARS-CoV-2 infection in NCI-H1299 cells was maintained by minute 246 traces of ACE2 expressed below the detection limit of our system, we blocked ACE2 by antibodies. 247

Individual incubation of Vero E6 and Calu-3 cells with three ACE2-neutralizing antibodies 248 abolished and diminished SARS-CoV-2 infection, respectively (Fig. 6F, Fig. S3A in spike plasmid-transfected cells . Of note, the latter study used plasmids 308 expressing spikes lacking 19 C-terminal amino acids, an experimental modification that has been 309 widely accepted for artificial enhancement of cell surface expression and lentiviral incorporation 310 of spike (Yu et al., 2021) , and that we refrained to adopt in order to maintain the expression context 311 as physiological as possible. The observation of reduced processing translated into lower levels 312 of lentivirus-associated spike, which may be the cause of the reduced infectivity of particles. Even 313 though impaired maturation did not detectably alter virion-associated spike levels under our 314 experimental conditions, we cannot exclude that it may still modulate the kinetics of virus particle 315 secretion and/or the quality of secreted SARS-CoV-2 particles. in NCI-H1299 cells was higher than for B.1.1.7, which would be expected given the observed 362 differences in epidemic growth rates for these VOCs. More work will be necessary to understand 363 the nature of the alternative entry mechanism, the identity of the entry-independent and variant-364 specific difference in replication as a function of spike, as well as the possibility that two VOCs 365 may have undergone convergent evolution towards utilization of this same unknown mechanism. 366 Also, whether the ACE2-independent entry is linked to the replication advantage, e.g. by 367 potentially more efficient virion release due to ACE2 scarcity or absence, remains unclear at this Codon-optimized, C-terminally tagged spike cDNAs in pCG were generated using pCG-SARS-416

CoV-2 spike Wuhan as a template (Hoffmann et al., 2020) Virus titrations, RNA extractions and RT-qPCR: To determine virus titers from 50 mg of 608 lung tissue, tissue homogenates were prepared using a bead mill (Analytic Jena) and 10-fold serial 609 dilutions were prepared in MEM, which were then added to Vero E6 cells in 12-well plates. The 610 dilutions were removed after two hours and cells were overlaid with 1.25% microcrystalline 611 cellulose (Avicel) in MEM supplemented with 10% FBS and penicillin/streptomycin. Two days 612 later, cells were formalin-fixed, stained with crystal violet, and plaques were counted. RNA was 613 extracted from 25 mg of lung homogenates and oropharyngeal swabs using the innuPREP Virus 614 RNA kit (Analytic Jena). Viral RNA copies were quantified in 10% of the obtained eluate volume 615 with a one-step RT-qPCR reaction using a standard curve and the Luna Universal Probe One-616

Step RT-qPCR kit (New England Biolabs) and previously published TaqMan primers and probe 617 To determine the processing and incorporation of spike from infected cells, cells and 708 purified virus particles were lysed with RIPA (ThermoFisher Scientific) buffer supplemented with 709 complete protease inhibitor cocktail (Roche) for 30 min at 4°C. Subsequently, lysates were 710 centrifuged for 15 min at 4°C and 15,000 rpm to remove cell debris. The supernatants were mixed 711 with 4x Laemmli buffer, which was supplemented with 10% beta-mercaptoethanol, and lysates 712 were boiled for ten minutes at 95°C to ensure protein denaturation and virus inactivation. Protein 713 concentration was determined by BCA protein assay (ThermoFisher Scientific) and 20 µg total 714 protein was loaded. Proteins were separated by SDS-PAGE on a 6% gel and transferred to a 715 nitrocellulose membrane (0.45 µm pore size, GE Healthcare) by Trans-Blot Turbo system 716 (BioRad). Membranes were blocked with 5% dried milk in 0. 

Comparing infectivity and 967 virulence of emerging SARS-CoV-2 variants in Syrian hamsters

Construction and use 969 of retroviral vectors encoding the toxic gene barnase

Neutralization of SARS-CoV-2 variants by 972 convalescent and BNT162b2 vaccinated serum

Increased transmission of SARS-CoV-2 975 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary 976 airway cells or antibody escape

Detection of 2019 novel coronavirus (2019-nCoV) 979 by real-time RT-PCR

CMMID COVID-19 Working Group

Increased mortality in community-tested cases of 982 SARS-CoV-2 lineage B.1.1.7

SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host 986 transcriptional profiles

Pharmacological Rescue of Conditionally 989 Reprogrammed Cystic Fibrosis Bronchial Epithelial Cells

The protein 992 expression profile of ACE2 in human tissues

Safety, reactogenicity, and immunogenicity 995 of homologous and heterologous prime-boost immunisation with ChAdOx1 nCoV-19 and 996 BNT162b2: a prospective cohort study

SARS-CoV-2 Cell Entry 999 Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

SARS-CoV-2 variant 1003 B.1.617 is resistant to bamlanivimab and evades antibodies induced by infection and 1004 vaccination

SARS-CoV-2 variants B.1.351 and B.1.1.248: 1007 Escape from therapeutic antibodies and antibodies induced by infection and vaccination

Estimating infectiousness 1010 throughout SARS-CoV-2 infection course

SARS-CoV-2 evolution during 1013 treatment of chronic infection

Densely sampled viral trajectories 1016 suggest longer duration of acute infection with B.1.1.7 variant relative to non-B

Viral dynamics of SARS-CoV-2 variants in 1020 vaccinated and unvaccinated individuals (medRxiv)

Antibody Protects from Lung Pathology in a COVID-19 Hamster Model

Human organoid systems reveal in vitro 1027 correlates of fitness for SARS-CoV

Delta spike P681R mutation enhances SARS-CoV-2 fitness 1030 over Alpha variant

Spike protein cleavage-activation mediated by the 1033 SARS-CoV-2 P681R mutation: a case-study from its first appearance in variant of interest

A.23.1 identified in Uganda. bioRxiv

Functional 1036 evaluation of proteolytic activation for the SARS-CoV-2 variant B

Recurrent emergence of SARS-CoV-2 spike deletion 1040 H69/V70 and its role in the Alpha variant B

SARS-CoV-2 B.1.617.2 Delta variant replication and 1043 immune evasion

Low dose inocula of SARS-CoV-2 Alpha variant transmits more 1046 efficiently than earlier variants in hamsters

SARS-CoV-2 Beta variant infection 1049 elicits potent lineage-specific and cross-reactive antibodies

SARS-CoV-2 spike L452R variant evades cellular immunity 1052 and increases infectivity

Infection of Syrian Hamster Does Not Cause More Severe Disease, and 1055 Naturally Acquired Immunity Confers Protection

Clinical and virological features of SARS-CoV-2 variants of 1058 concern: a retrospective cohort study comparing B.1.1.7 (Alpha), B.1.315 (Beta), and B.1.617.2 1059 (Delta)

Tracking the international spread of SARS-CoV-2 1062 lineages B.1.1.7 and B.1.351/501Y-V2

NCI-Navy Medical 1068 Oncology Branch cell line data base

Reduced sensitivity of SARS-CoV-2 variant 1071 Delta to antibody neutralization

Systematic analysis of SARS-CoV-2 1074 infection of an ACE2-negative human airway cell

SARS-CoV-2 Alpha, Beta and Delta variants 1077 display enhanced Spike-mediated Syncytia Formation

SARS-CoV-2 B.1.1.7 and 1079 B.1.351 spike variants bind human ACE2 with increased affinity

Comparison of potency assays to assess 1082 SARS-CoV-2 neutralizing antibody capacity in COVID-19 convalescent plasma

UK B.1.1.7 variant exhibits increased 1086 respiratory replication and shedding in nonhuman primates

Long-term expansion of epithelial 1089 organoids from human colon, adenoma, adenocarcinoma

Disease Severity, Fever, Age, and Sex Correlate With 1093 SARS-CoV-2 Neutralizing Antibody Responses

SARS-CoV-2 variant B.1.1.7 is susceptible to neutralizing 1096 antibodies elicited by ancestral Spike vaccines

Reduced neutralization of SARS-CoV-2

1.1.7 variant by convalescent and vaccine sera

Clinical and 1102 virological characteristics of hospitalised COVID-19 patients in a German tertiary care centre 1103 during the first wave of the SARS-CoV-2 pandemic: a prospective observational study

Evolution of enhanced innate immune evasion by the SARS-CoV-2 B

Replicative Fitness of a SARS-CoV-2 20I/501Y.V1 Variant 1110 from Lineage B.1.1.7 in Human Reconstituted Bronchial Epithelium

The Roborovski Dwarf Hamster Is A Highly 1113 Susceptible Model for a Rapid and Fatal Course of SARS-CoV-2 Infection

Enhanced fitness of SARS-CoV-2 variant of concern B.1.1.7, 1117 but not B.1.351, in animal models

Assessing transmissibility of SARS-CoV-2 1120 lineage B.1.1.7 in England

SARS 1122 coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic 1123 pathway

Transmission, viral kinetics and clinical characteristics of the emergent SARS-CoV

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Virological assessment of hospitalized patients 1132 with COVID-2019

Effect of SARS-CoV-2 B.1.1.7 mutations on spike protein 1135 structure and function

Three-Dimensional Human Alveolar Stem Cell Culture Models Reveal 1138 Infection Response to SARS-CoV-2

Effects of a major deletion in the SARS-CoV-2 1141 genome on the severity of infection and the inflammatory response: an observational cohort 1142 study

Deletion of the SARS-CoV-2 Spike Cytoplasmic Tail 1145 Increases Infectivity in Pseudovirus Neutralization Assays

Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo

Post-entry, spike-dependent replication advantage of B.1.1.7 and B.1.617.2 over B

SARS-CoV-2 in an ACE2-deficient human lung cell line

7 spike is not superior in mediating entry compared to B.1 spike (A) Calu-3 cells were transduced for 72 hours with increasing amounts of lentiviral particles (0.1 µl, 1 µl and 10 µl) pseudotyped with either WT-or B.1.1.7-spike proteins

µM Camostat (TMPRSS2 inhibitor) or 15 µM CMK (furin inhibitor), infected and entry efficiency was determined by sgN quantitative RT-PCR. DMSO: Dimethylsulfoxid, CatL: Cathepsin L, PS: PitStop, Cam: Camostat mesylate Supplemental Movies 1 and 2

Onset of CPE was monitored by live cell imaging until 70 hours post infection