SARS-CoV-2 Omicron spike mediated immune escape and tropism shift

Abstract

The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and is characterised by multiple spike mutations across all spike domains. Here we show that Omicron BA.1 has higher affinity for ACE2 compared to Delta, and confers very significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesevir and molnupiravir retain efficacy against Omicron BA.1. We found that in human nasal epithelial 3D cultures replication was similar for both Omicron and Delta. However, in lower airway organoids, Calu-3 lung cells and gut adenocarcinoma cell lines live Omicron virus demonstrated significantly lower replication in comparison to Delta. We noted that despite presence of mutations predicted to favour spike S1/S2 cleavage, the spike protein is less efficiently cleaved in live Omicron virions compared to Delta virions. We mapped the replication differences between the variants to entry efficiency using spike pseudotyped virus (PV) entry assays. The defect for Omicron PV in specific cell types correlated with higher cellular RNA expression of TMPRSS2, and accordingly knock down of TMPRSS2 impacted Delta entry to a greater extent as compared to Omicron. Furthermore, drug inhibitors targeting specific entry pathways demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that mediates cell entry via plasma membrane fusion. Instead, we demonstrate that Omicron spike has greater dependency on cell entry via the endocytic pathway requiring the activity of endosomal cathepsins to cleave spike. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was dramatically impaired compared to the Delta spike. Overall, Omicron appears to have gained significant evasion from neutralising antibodies whilst maintaining sensitivity to antiviral drugs targeting the polymerase. Omicron has shifted cellular tropism away from TMPRSS2 expressing cells that are enriched in cells found in the lower respiratory and GI tracts, with implications for altered pathogenesis.

Figure 1.. Sensitivity of SARS-CoV-2 Omicron to clinically approved monoclonal antibodies directed against spike and to vaccine elicited neutralizing antibodies.

a. Side Surface representation of the Omicron spike protein. b. Top down surface representation of the Omicron spike. Spike homotrimer structures were created predicted in silico by the Alphafold2 software package. Individual mutations making up the Omicron spike are highlighted in red on each of the three homotrimers. c. Predicted interaction sites for REGN 10933 and 10897 monoclonal antibodies with Omicron spike RBD. Hydrogen bonds are indicated with dashed lines. Predicted contact points are shown as spherical representation. Omicron mutations labelled. d. titration of monoclonal antibodies REGN 10933 and 10897 and combination against replication competent Delta and Omicron viruses. NT: Neutralising titre. e.Neutralisation of spike pseudotyped virus by sera from vaccinated individuals over three time points following dose two (ChAdOx-1 or BNT162b2) and dose three (BNT162b2 only) e. n=20 ChAdOx-1 or f. n=20 BNT12b2. GMT (geometric mean titre) with s.d are presented. Data representative of two independent experiments each with two technical replicates. **p<0.01, ***p<0.001, ****p<0.0001 Wilcoxon matched-pairs signed rank test, ns not significant.

Figure 2:. SARS-CoV-2 Omicron and Delta Variant live virus replication in 3D tissue culture systems and 2D cell lines.

a. overview of viruses and culture systems used hNEC (human nasal epithelial cultures). b-d. Spreading infection by replication competent Omicron versus Delta variant over time in b. hNEC c. Calu-3 d. Caco-2 Npro e. HeLa-A2T2. Viral RNA and/or infectious virus in supernatant (TCID50) were measured f-g. Western blot of f. Two live Omicron and one Delta virus isolates probed with antibodies to S2, S1 and NP with quantification of S2 and S1 to total spike ratio, g. Vero E6 A2T2 producer cell lysates infected with live isolates probed with antibodies to S2, S1 and NP. Housekeeping gene GAPDH as loading control. Data are representative of two independent experiments, h-j. Subcellular localisation of Spike in SARS-Cov-2 Delta vs Omicron infected cells. Subcellular distribution of Omicron. Delta Spike proteins in Hela-ACE2 cells infected with live virus isolates, h. Cells on coverslips were infected for 24 h. fixed and stained with anti-Spike, anti-GM130-cis-Golgi, phalloidin 647 and DAPI, and imaged on a Leica TCS SP8 confocal microscope. i. The distance of spike proteins from nucleus at 24 hpi. j. Quantitation of Spike-Golgi colocalisation in infected cells. Values were calculated using Pearson’s correlation coefficient. Scale bars: 10 μm and 1 μm, respectively. * p<0.05; NS – not significant

Figure 3:. SARS-CoV-2 Omicron Variant spike enters ceils less efficiently by TMPRSS2 mediated plasma membrane fusion.

. a. Graphical representation of Omicron spike mutations present in expression plasmid used to generate lentiviral pseudotyped virus (PV). Mutations coloured according to location in spike; bold mutations are novel to this lineage and have not been identified in previous variants of concern (VOCs). b. PV entry in airway organoids, Calu-3 lung cells, gall bladded organoids, HI299 lung cells, HeLa-ACE2 overexpressing cells and HEK 293-ACE2 overexpressing cells. Black – WT Wuhan-1 D614G, Blue – Delta B.1.617.2, Green Omicron BA.1, c. mRNA transcripts for ACE2 and TMPRSS2 in indicated cell types and organoids as measured by qPCR. Samples were run in quadruplicate, d. Entry of PV expressing spike in 293T cells transduced to overexpress ACE2 and (i) depleted for TMPRSS2 (A2Δ T2) or (ii) overexpressing TMPRSS2 (A2T2). e. Entry of PV expressing spike in 293T cclls with endogenous (−) or overexpressed TMPRSS2 (T2). f. illustration of two cell entry pathways known to be used by SARS-CoV-2. g. Titration of inhibitors in A549-ACE2-TMPRSS2 (A549A2T2) cclls using PV expressing Delta (orange) or Omicron (grey) spike in the presence of the indicated doses of Camostat or E64d, then analysed after 48 hours. % inhibition was calculated relative to the maximum luminescent signal for each condition. For each variant and dilution, mean ± SEM is shown for an experiment conducted in duplicate. Data are representative of two independent experiments h. Titration of inhibitors in A549-ACE2-TMPRSS2 (A549A2T2)-based luminescent reporter cells using live virus. Cells were infected at MOI = 0.01 with Delta (orange) or Omicron (grey) variants in the presence of the indicated doses of Camostat or E64d, then analysed after 24 hours. % inhibition was calculated relative to the maximum luminescent signal for each condition. For each variant and dilution, mean ± SEM is shown for an experiment conducted in triplicate, i. ACE2 and TMPRSS2 mRNA expression by qPCR in human lung tissue (4 pieces of tissue each from upper and lower). Lower – Lung parenchyma Upper - main bronchus. One tissue sample from each region was tested in quadruplicate. *p<0.05, **p<0.01, ****p<0.0001. Data are representative of two independent experiments

Figure 4:. SARS-CoV-2 Omicron variant spike shows impaired cell-cell fusion activity and smaller infection foci generated by live virus.

a. Schematic of cell-cell fusion assay. b. spike expression at the cell surface as determined by flow cytometry, showing % positive cells c. Reconstructed images at 16 hours of GFP+ syncytia. d. Quantification of cell-cell fusion kinetics showing percentage of green area to total cell area over time (WT is Wuhan-1 D614G). Mean is plotted with error bars representing SEM. Data are representative of at least two independent experiments, e. Left: representative images of H1299-ACE2 cell monolayers infected either with the live Delta (top 3 wells) or Omicron (bottom 3 wells) in a semi-solid media inhibitory for cell free infection. Cells were fixed 18–20 hours post-infection and stained for SARS-CoV-2 spike to visualize infection foci. Bar is 2mm. Middle and right panels: Quantified focus numbers and foci area (geometric mean and geometric std) for Omicron and Delta live virus infections. Data are from n=112 Delta infected wells and n=111 of Omicron infected wells from five independent experiments. ** p<0.0001 by the Wilcoxon sign-rank test.