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

Abstract

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)¹. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

Fig. 1. Rapid expansion of Delta variant B.1.617.2 cases in India and reduced sensitivity to neutralizing antibodies from sera derived following infection and vaccination.

a, Proportion of lineages in incident cases of SARS-CoV-2 in India 2020–2021. b, Surface representation of the SARS-CoV-2 B.1.671.2 spike trimer (PDB: 6ZGE). Red, L19R; green, del157/158; blue, L452R; yellow, T478K. The white dotted box indicates the location of the D950N substitution (orange). c, Neutralization of the Delta variant by convalescent human serum from mid-2020. Fold change in serum neutralization of 100 TCID₅₀ of B.1.17 (Alpha), B.1.351 (Beta) and B.1617.2 (Delta) variants relative to WT (IC19); n = 12. Shown is the ID₅₀, the serum dilution required for 50% virus inhibition, expressed as GMT (from technical replicates) with s.d. d, Neutralization of B.1617.2 live virus by sera from vaccinated individuals (n = 10 ChAdOx1 or n = 10 BNT12b2), compared with B.1.1.7 and Wuhan-1 WT. The graph presents the average of two independent experiments. e, Neutralization of B.1.617 spike PV and WT (Wuhan-1 D614G) by vaccine sera (n = 33 ChAdOx1 or n = 32 BNT162b2). The data are representative of two independent experiments each with two technical replicates. *P < 0.05, **P < 0.01, ****P < 0.0001 (Wilcoxon matched-pairs signed rank test); NS, not significant.

Fig. 2. Delta variant live virus replication kinetics and spike-mediated infectivity.

a–d, Live virus replication comparing B.1.1.7 with B.1.617.2. Calu-3 cells were infected with variants at an MOI of 0.1. a, Viral loads measured by qPCR in cell lysates. b, Viral protein levels in cell lysates. c, d, Live virus produced from infected Calu-3 cell supernatants was collected and used to infect permissive Vero E6 ACE2/TMPRSS2 cells to measure viral loads (c) or TCID₅₀ ml⁻¹ (d). e, f, Virus replication kinetics in the HAE system. g, Live virus replication in airway epithelial organoid cultures. Airway epithelial organoids were infected with the SARS-CoV-2 variants B.1.1.7 and B.1.617.2 at an MOI of 1. Cells were lysed and total RNA was isolated. qPCR was used to determine the number of copies of the nucleoprotein gene in cells and the infectivity of cell-free virus measured by infection of Vero E6 ACE2/TMPRSS2 cells. The data are representative of two independent experiments. dpi, days post-infection. h, i, Western blots of PV virions (h) and cell lysates (i) of 293T producer cells following transfection with plasmids expressing lentiviral vectors and SARS-CoV-2 S B.1.617.1 and Delta variant B.1.617.2 versus WT (Wuhan-1 with D614G), probed with antibodies to HIV-1 p24 and SARS-Cov-2 S2. j, Calu-3 cell entry by spike B.1.617.2 and B.1.617.1 versus WT D614G parental plasmid PVs. The data are representative of three independent experiments. k, Growth kinetics of B.1.617.1 and B.1.617.2 variants. Viral isolates of B.1.617.1 and B.1.617.2 were inoculated into Calu-3 cells, and viral RNA in the culture supernatant was quantified by real-time RT–PCR. The TCID₅₀ of released virus in supernatant was measured over time. Assays were performed in quadruplicate. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The data are representative of two independent experiments. Uninfected cells are represented by a minus symbol. NP, nucleocapsid protein.

Fig. 3. SARS-CoV-2 B.1.617.2 infection in vaccinated HCWs.

a–c, Maximum-likelihood phylogenies of vaccine breakthrough SARS-CoV-2 sequences among vaccinated HCWs at three centres. Phylogenies were inferred with IQTREE2 with 1,000 bootstrap replicates. SNPs, single nucleotide polymorphisms.

Extended Data Fig. 1. Delta variant B.1.617.2 shows reduced sensitivity to monoclonal antibodies.

Neutralisation by a panel of NTD- and RBD-specific mAbs against WT and B.1.617.2 mutant SARS-CoV-2 pseudotyped viruses. a. Neutralisation of WT D614 (black) and B.1.617.2 mutant (blue) pseudotyped SARS-CoV-2-VSV by 6 selected mAbs from one representative experiment out of 2 independent experiments. S2X333 is an NTD-specific mAb, S2D97, S2E12 and S2X58 are RBM-specific mAbs, while S2X35 and S2X305 are non-RBM mAbs. b. Neutralisation of WT and B.1.617.2 VSV by 38 mAbs targeting NTD (n = 3), RBM (n = 26, including 5 clinical stage mAb) and non-RBM (n = 9). Shown are the mean IC50 values (ng/ml) from 2 independent experiments. Non-neutralising IC50 titers were set at 10⁴ ng/ml. c. Neutralisation shown as mean IC50 values (upper panel) and average fold change of B.1.617.2 relative to WT (lower panel) of 38 mAbs tested in 2 independent experiments (including 5 clinical-stage mAbs), tested using Vero E6 cells expressing TMPRSS2. d–e, Neutralisation of WT D614 (black) and B.1.617.2 mutant (blue/red) pseudotyped SARS-CoV-2-VSV by 5 clinical-stage mAbs using Vero E6 cells expressing TMPRSS2 (d) or not (e). Shown is one representative experiment out of 2 independent experiments.

Extended Data Fig. 2. Spike cleavage in B.1.617.2 virions compared to B.1.1.7. and spike mediated cell-cell fusion.

a. Representative western blot analysis of spike and nucleoprotein present in SARS-CoV-2 particles from the indicated viruses produced in Vero E6 ACE2/TMPRSS2 cells 48 h post infection. The arrowhead identifies the S2 subunit. b. Quantification of cleaved and full-length spike of the indicated viruses. c. Schematic of cell-cell fusion assay. d. Reconstructed images at 10 h of GFP positive syncytia formation. Scale bars represent 400 mm. e. western blot of cell lysates 48 h after transfection of spike plasmids.f,g. Quantification of cell-cell fusion kinetics showing percentage of green area to total cell area over time. Mean is plotted with error bars representing SEM. h. Comparison of impact of post vaccine sera (n = 2) on PV neutralisation (top) and cell-cell fusion (bottom), comparing WT and Delta variant B.1.671.2. Data are representative of at least two independent experiments.

Extended Data Fig. 3. B.1.617.2 spike confers increased cell entry.

a. diagram showing mutations present in spike plasmids used for cell entry PV experiments b. Single round infectivity on different cell targets by spike B.1.617.1 and B.1.617.1 versus WT (Wuhan-1 D614G) PV produced in 293T cells. Data are representative of three independent experiments. Statistics were performed using unpaired Student t test. c. Western blotting of supernatants from transfected 293T probing for S2 and p24 in PV and showing no spike control.

Extended Data Fig. 4. Breakthrough SARS-CoV-2 infections amongst vaccinated health care workers (HCW).

a. Case frequencies of five most commonly occurring SARS CoV-2 lineages over a six week period from March to April 2021 for Delhi b,c,d. case frequency graph for hospital 1, 2 and 3 respectively by date of testing. e. Comparison of IgG antibody titres between a control group of vaccinated individuals receiving two doses of ChadOx-1 who have not been infected with SARS-CoV-2, with vaccinated healthcare workers who had received two doses and subsequently tested positive for SARS-CoV-2. f. Ct values in nose/throat swabs from HCW testing positive by hospital. Bars represent Mean and 95% CI. Ct values were compared using the Student t test.