SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha

Characterization of intrinsic and effective fitness changes caused by temporarily fixed mutations in the SARS-CoV-2 spike E484 epitope and identification of an epistatic precondition for the evolution of E484A in variant Omicron

Acknowledgements: We gratefully acknowledge all data contributors, i.e., the authors and their originating laboratories responsible for obtaining the specimens, and their submitting laboratories for generating the genetic sequence and metadata and sharing via the GISAID Initiative, on which this research is partly based.Funder: Charité - Universitätsmedizin Berlin (3093)Abstract Background Intrinsic fitness costs are likely to have guided the selection of lineage-determining mutations during emergence of variants of SARS-CoV-2. Whereas changes in receptor affinity and antibody neutralization have been thoroughly mapped for individual mutations in spike, their influence on intrinsic replicative fitness remains understudied. Methods We analyzed mutations in immunodominant spike epitope E484 that became temporarily fixed over the pandemic. We engineered the resulting immune escape mutations E484K, -A, and -Q in recombinant SARS-CoV-2. We characterized viral replication, entry, and competitive fitness with and without immune serum from humans with defined exposure/vaccination history and hamsters monospecifically infected with the E484K variant. We additionally engineered a virus containing the Omicron signature mutations N501Y and Q498R that were predicted to epistatically enhance receptor binding. Results Multistep growth kinetics in Vero-, Calu-3, and NCI-H1299 were identical between viruses. Synchronized entry experiments based on cold absorption and temperature shift identified only an insignificant trend toward faster entry of the E484K variant. Competitive passage experiments revealed clear replicative fitness differences. In absence of immune serum, E484A and E484Q, but not E484K, were replaced by wildtype (WT) in competition assays. In presence of immune serum, all three mutants outcompeted WT. Decreased E484A fitness levels were over-compensated for by N501Y and Q498R, identifying a putative Omicron founder background that exceeds the intrinsic and effective fitness of WT and matches that of E484K. Critically, the E484A/Q498R/N501Y mutant and E484K have equal fitness also in presence of pre-Omicron vaccinee serum, whereas the fitness gain by E484K is lost in the presence of serum raised against the E484K variant in hamsters. Conclusions The emergence of E484A and E484Q prior to widespread population immunity may have been limited by fitness costs. In populations already exposed to the early immune escape epitope E484K, the Omicron founder background may have provided a basis for alternative immune escape evolution via E484A. Studies of major antigenic epitope changes with and without their epistatic context help reconstruct the sequential adjustments of intrinsic fitness versus neutralization escape during the evolution of major SARS-CoV-2 variants in an increasingly immune human population. </jats:sec

Absence of detectable fitness advantages of VOC Alpha in primary human respiratory cells, organoids, and hamsters.

(A) Virus growth kinetics were performed in infected hNAECs (MOI 0.1). Samples were collected from the apical and basal side at indicated time points and titrated by plaque assay. n = 3 biological replicates. (B) Virus growth kinetics was conducted in infected bronchial AEC (MOI 0.5). Samples were collected from the apical side and titrated by plaque assay. Data are derived from 1 experiment conducted in triplicates. (C) Intestinal organoids were infected (MOI 0.05) and viral load in supernatant (left) and organoid lysates (right) was quantified at indicated time points by E-gene-specific quantitative RT-PCR. Data are derived from 4 independent experiments. (D) Virus replication was monitored in infected lung organoids (MOI 1). Samples harvested at indicated time points were titrated by plaque assay. Data are derived from 3 independent experiments. (E) Dwarf hamsters were intranasally infected (100,000 PFU) and infectious virus particles from lung homogenates were quantified using plaque assay (left). Donor hamsters were cohoused with naive animals and transmission efficiency was determined from lung homogenates at the indicated time points (right). n = 1–3 animals per experimental condition. Dotted horizontal lines indicate the lower detection limit of the plaque assays. AEC, airway epithelial culture; GE, genome equivalents; hNAEC, human nasal airway epithelial culture; MOI, multiplicity of infection; n.d., not detected; PFU, plaque-forming units; RT-PCR, real-time PCR; VOC, variant of concern. See S1 Data.</p

Enhanced cell–cell fusion and reduced virus particle entry by VOC Alpha SARS-CoV-2 spike.

(A) For Tat-mediated cell–cell fusion assay, CHO cells were cotransfected with plasmids expressing indicated spike-HA and HIV-1 Tat. LTR-luciferase-expressing target TZM-bl cells were transfected with plasmids encoding human ACE2 and TMPRSS2. Transfected cells were cocultured for 8 hours and luciferase expression resulting from intercellular Tat transfer was quantified luminometrically. All values were normalized to B.1 spike (indicated by a dotted line). Shown are results from 3–6 biological replicates, each performed in triplicates. (B) Calu-3 cells were transduced with lentiviral pseudoparticles expressing luciferase and decorated with indicated spike-HA. Transduction efficiency was quantified luminometrically. Dotted line indicates background levels of luciferase nontransduced cultures. Shown are results from 6 independent biological replicates (using independent lentivirus particle preparations), each performed in triplicates, indicated by symbols. (C) Indicated A549 cells were transduced with increasing quantities (0.5 μl, 5 μl, and 50 μl) of lentiviral, luciferase-expressing particles pseudotyped with B.1- or VOC Alpha-spike. Transduction efficiency was determined luminometrically. Dotted line indicates luciferase background level of luciferase detected in nontransduced cells. Symbols represent individual values of 3 biological replicates, each performed in triplicates. (D, E) Indicated A549 (D) and Calu-3 (E) cells were infected at 4°C with B.1 or VOC Alpha isolates (MOI 1) to allow synchronized infection. Total cellular RNA was isolated at the indicated time points and nucleocapsid-encoding sgRNA was quantified by Q-RT-PCR. N = mean of 3–4 biological replicates, indicated by symbols. (F) Synchronized infection of Calu-3 cells was performed with B.1 and VOC Alpha virions that were pretreated with trypsin for 1 hour at 37°C. Data were normalized to the respective log10 relative N sgRNA level of the untreated (0 μg/ml) 4 hours postinfection sample (dotted line). Means +/− SD of 6 independently performed experiments are shown. (G) Synchronized infection of Calu-3 cells was performed with B.1 and VOC Alpha virions that were pretreated with saliva (pooled saliva from 3 healthy donors) for 1 hour at 37°C. Data were normalized to the respective log10 relative sgRNA N level of the untreated (-) 4 hours postinfection sample (dotted line). Results show means of 4 independently performed experiments, which were each performed in triplicates. Del, deletion; MOI, multiplicity of infection; Q-RT-PCR, quantitative real-time PCR; RLU, relative light units; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA N, subgenomic nucleocapsid RNA; TMPRSS2, transmembrane protease serine subtype 2; VOC, variant of concern. See S1 Data.</p

VOC Alpha and B.1 efficiently dampen induction of innate immunity in hBAECs.

hBAECs were infected with B.1 or VOC Alpha (MOI of 0.5) and cell lysates were generated at the indicated time points followed by total RNA extraction. The experiment was performed with cells derived from 1–5 adult donors and that were infected in duplicates. (A) Cell-associated expression of envelope in hBAECs during the early phase of infection determined by Q-RT-PCR. TBP was used for normalization. (B) Cell-associated expression of sgN in hBAECs during an early phase of infection determined by Q-RT-PCR. (C–I) Expression of the indicated genes was determined by Q-RT-PCR. Shown is the mean fold change +/− SD. (J) Relative change (to preinfection) of cytokines and chemokines concentration in the basal medium of infected hBAECs (MOI 0.5). Concentration of cytokines and chemokines was determined by MagPix Luminex technology. Paired t tests were conducted between B.1 and VOC Alpha-infected groups and scored negative. AEC, airway epithelial cells; hBAEC, human bronchial airway epithelial cell; MOI, multiplicity of infection; Q-RT-PCR, quantitative real-time PCR; sgN, subgenomic nucleocapsid; TBP, TATA-binding protein; VOC, variant of concern. See S1 Data.</p

Primers used for reconstruction of recombinant SARS-CoV-2.

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Primers used for quantitative RT-PCR.

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Similar abundance of sgN RNAs, genome replication, and low but similar expression of IFNs, proinflammatory cytokines, and ISGs in B.1 and VOC Alpha-infected H1299 cells.

NCI-H1299 cells were infected with B.1 or VOC Alpha (MOI of 2), and viral replication, viral transcription, and expression of innate immune genes were determined by Q-RT-PCR from cell lysates at 24 and 48 hours postinfection. (A) Expression of cell-associated envelope. (B) Expression of cell-associated sgN RNA. TBP was used for normalization. (C) Expression of the indicated genes was determined by specific Q-RT-PCR. TBP was used for normalization. Shown is the mean fold change +/− SD of 3 biologically independent experiments that were each conducted in quadruples. RVFV cl.13, which is devoid of its IFN antagonist NSs, was included for the expression of IFNs, ISGs, and pro-inflammatory cytokines. GE, genome equivalents; IFN, interferon; ISG, IFN-stimulated gene; Q-RT-PCR, quantitative real-time PCR; RVFV cl.13, Rift Valley Fever Virus clone 13; sgN, subgenomic nucleocapsid; TBP, TATA-binding protein. See S1 Data. (TIF)</p