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

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.

is known to be more transmissible than previous versions of the virus bearing the D614G spike substitution, while maintaining sensitivity to vaccine-elicited neutralizing antibodies 2,3 . The B.1.617 variant was first identified in the state of Maharashtra in late 2020 4 , spreading throughout India and to at least 90 countries.

Delta variant and neutralizing antibodies
We first plotted the relative proportion of variants in new cases of SARS-CoV-2 in India since the start of 2021. Although B.1.617.1 emerged earlier, the Delta variant B.1.617.2 has become more dominant (Fig. 1a). We hypothesized that B.1.617.2 would exhibit immune evasion to antibody responses generated by previous SARS-CoV-2 infection. We used sera from 12 individuals infected during the first UK wave in mid-2020. These sera were tested for their ability to neutralize a B.1.617.2 viral isolate, in comparison with a B.1.1.7 variant isolate and a wild-type (WT) Wuhan-1 virus bearing D614G in spike. The Delta variant contains several spike alterations that are located at positions within the structure that are predicted to alter its function (Fig. 1b). We found that the B.1.1.7 virus isolate was 2.3-fold less sensitive to the sera than the WT, and that B.1.617.2 was 5.7-fold less sensitive to the sera (Fig. 1c). Importantly, in the same assay, the B.1.351 Beta variant that was first identified in South Africa demonstrated an 8.2-fold loss of neutralization sensitivity relative to the WT.
We used the same B.1.617.2 live virus isolate to test susceptibility to vaccine-elicited serum neutralizing antibodies in individuals following vaccination with two doses of ChAdOx1 or BNT162b2. These experiments showed a loss of sensitivity for B.1.617.2 compared with WT Wuhan-1 bearing D614G of around eightfold for both sets of vaccine sera and reduction against B.1.1.7 that did not reach statistical significance (Fig. 1d). We also used a pseudotyped virus (PV) system to test the neutralization potency of a larger panel of 65 vaccine-elicited sera, this time against B.1.617.1 as well as B.1.617.2 spike compared with Wuhan-1 D614G spike (Fig. 1e). Comparison of demographic data for vaccinees showed similar characteristics (Extended Data Table 1). The mean geometric mean titre (GMT) against Delta variant spike PV was lower for ChAdOx1 than for BNT162b2 (GMT 654 versus 3,372, P < 0001, Extended Data Table 1).
We investigated the role of the B.1.617.2 spike as an escape mechanism by testing 33 spike-specific monoclonal antibodies with an in vitro PV neutralization assay using Vero E6 target cells expressing transmembrane protease serine 2 (TMPRSS2) and the Wuhan-1 D614G SARS-CoV-2   Fig. 1).

B.1.617.2 spike-mediated cell fusion
The plasma membrane route of entry, and indeed transmissibility in animal models, is critically dependent on the polybasic cleavage site between S1 and S2 (refs. 9,11,12 ) and cleavage of spike before virion release from producer cells. Alterations at P681 in the polybasic cleavage site have been observed in multiple SARS-CoV-2 lineages, most notably in the B.1.1.7 Alpha variant 13 . We previously showed that B.1.1.7 spike, bearing P681H, had significantly higher fusogenic potential than a D614G Wuhan-1 virus 13 . Here we tested B.1.617.1 and B.1.617.2 spike using a split GFP system to monitor cell-cell fusion (Extended Data Fig. 2c-g). The B.1.617.1 and B.1.617.2 spike proteins mediated higher fusion activity and syncytium formation than WT, probably mediated by P681R (Extended Data Fig. 2f, g). We next titrated sera from ChAdOx1 vaccinees and showed that indeed the cell-cell fusion could be inhibited in a manner that mirrored the neutralization activity of the sera against PV infection

B.1.617.2 vaccine breakthrough infection
We hypothesized that vaccine effectiveness against B.1.617.2 would be compromised relative to that against other circulating variants.  (Fig. 3a). Phylogenetic analysis demonstrated a group of highly related, and in some cases, genetically indistinct sequences that were sampled within 1 or 2 days of each other ( Fig. 3a and Extended Data Fig. 4b). We next looked in greater detail at the vaccination history of affected individuals. Nearly all had received two doses at least 21 days previously. We obtained similar data on vaccine breakthrough infections in two other health facilities in Delhi with 1,100 and 4,000 HCW staff members, respectively (Fig. 3b, c and Extended Data Fig. 4c, d). In hospital 2, there were 118 sequences, representing more than 10% of the workforce over a 4-week period. After filtering, we reconstructed phylogenies using 66 with high-quality whole-genome coverage >95%. In hospital 3, there were 70 symptomatic infections, with 52 high-quality genomes used for inferring phylogenies after filtering.
Across the three centres, we noted that the median age and duration of infection of those infected with B.  Table 4).

Discussion
Here we have combined in vitro experimentation and molecular epidemiology to propose that increased replication fitness and reduced sensitivity of SARS-CoV-2 B.1.617.2 to neutralizing antibodies have contributed to the recent rapid increase of B.1.617.2, compared with B.1.1.7 and other lineages such as B.1.617.1, despite high vaccination rates in adults and/ or high prevalence of prior infection 16 . These data are consistent with modelling analyses that support combination of immune evasion and higher transmissibility as likely drivers of the increase in Delta in Delhi 17 .
We demonstrate evasion of neutralizing antibodies by a B.1.617.2 live virus with sera from convalescent patients, as well as sera from individuals vaccinated with two different vaccines, one based on an adenovirus vector (ChAdOx1) and the other mRNA based (BNT162b2). The reduced efficacy for imedevimab against B.1.617.2 shown here could translate to compromised clinical efficacy or possible selection of escape variants where there is immune compromise and chronic SARS-CoV-2 infection with B.1.617.2 (ref. 18 ).
It is important to consider that increased infectivity at mucosal surfaces and cell-cell fusion and spread 19 may also facilitate 'evasion' from antibodies 20 . Indeed, our work also shows that B.1.617.2 had a fitness advantage compared with B.1.1.7 across physiologically relevant systems including HAE and three-dimensional airway organoids 10 where cell-free and cell-cell infection are likely to be occurring together. These data support the notion of higher infectiousness of B.1.617.2, either due to higher viral burden or higher particle infectivity, resulting in higher probability of person-to-person transmission. We noted that B.1.617.2 live virus particles contained a higher proportion of cleaved spike than B.1.1.7, and postulated that this is involved in the mechanism of increased infectivity. This hypothesis was supported by our observation that PV particles bearing B.1.617.2 spike demonstrated significantly enhanced entry into a range of target cells.
Finally, we report ChAdOx1 vaccine breakthrough infections in HCWs at three Delhi hospitals, demonstrating reduced vaccine effectiveness against B.1.617.2. Therefore, strategies to boost vaccine responses against variants are warranted and attention to infection control procedures is needed in the post-vaccination era.

Online content
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Sequencing quality control and phylogenetic analysis
Three sets of fasta consensus sequences were obtained from three separate hospitals in Delhi, India. Initially, all sequences were concatenated into a multi-fasta file and then aligned to the reference strain MN908947.3 (Wuhan-Hu-1) with mafft v4.487 (ref. 21 ) using the --keeplength and --addfragments options. Following this, all sequences were passed through Nextclade v0.15 (https://clades.nextstrain.org/) to determine the number of gap regions. This was noted and all sequences were assigned a lineage with Pangolin v3.1.5 (ref. 22 ) and pangoLEARN (dated 15 June 2021). Sequences that could not be assigned a lineage were discarded. After assigning lineages, all sequences with more than 5% N regions were also excluded.
Phylogenies were inferred using maximum likelihood in IQTREE v2.1.4 (ref. 23 ) using a GTR + R6 model with 1,000 rapid bootstraps. The inferred phylogenies were annotated in R v4.1.0 using ggtree v3.0.2 (ref. 24 ) and rooted on the SARS-CoV-2 reference sequence (MN908947.3). Nodes were arranged in descending order and lineages were annotated on the phylogeny as coloured tips, alongside a heatmap defining the number of ChAdOx1 vaccine doses received by each patient. The final regression model was adjusted for age as a continuous variable, and sex and hospital as categorical variables. Model sensitivity and robustness to inclusion of these covariates was tested by an iterative process of sequentially adding the covariates to the model and examining the impact on the odd ratios and confidence intervals until the final model was constructed (Extended Data Table 4). The R 2 measure, as proposed by McFadden 26 , was used to test the fit of different specifications of the same model regression. This is was performed by sequential addition of the variables adjusted for including age, sex and hospital until the final model was constructed. In addition, the absolute difference in the Bayesian information criterion was estimated. The McFadden R 2 measure of final model fitness was 0.11, indicating reasonable model fit. The addition of age, sex and hospital in the final regression model improved the measured fitness. However, the absolute difference in the Bayesian information criterion was 13.34 between the full model and the model excluding the adjusting variable, providing strong support for the parsimonious model. The fully adjusted model was nonetheless used as the final model as the sensitivity analyses (Extended Data Table 4) showed robustness to the addition of the covariates.

Structural analyses
Neutralization titre analyses. The neutralization by vaccine-elicited antibodies after two doses of the BNT162b2 or ChadOx1 vaccines was determined by infections in the presence of serial dilutions of sera as described below. The ID 50 for each group was summarized as a GMT, and statistical comparisons between groups were performed with Mann-Whitney or Wilcoxon ranked sign tests. Statistical analyses were performed using Stata v13 and Prism v9.

PV experiments
Cells. HEK 293T CRL-3216, HeLa-ACE2 (gift from James Voss) and Vero CCL-81 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U ml −1 penicillin and 100 mg ml −1 streptomycin. All cells were regularly tested and found to be mycoplasma free. H1299 cells were a gift from Simon Cook. Calu-3 cells were a gift from Paul Lehner. A549 ACE2/TMPRSS2 (ref. 27 ) cells were a gift from Massimo Palmerini. Vero E6 ACE2/TMPRSS2 cells were a gift from Emma Thomson.
PV preparation for testing against vaccine-elicited antibodies and cell entry. Plasmids encoding the spike protein of SARS-CoV-2 D614 with a carboxy-terminal 19-amino-acid deletion with D614G were used. Mutations were introduced using the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent) following the manufacturer's instructions. Preparation of the B.1.1.7 S-expressing plasmid was described previously, but in brief, it was generated by stepwise mutagenesis. Viral vectors were prepared by transfection of 293T cells by using Fugene HD transfection reagent (Promega). 293T cells were transfected with a mixture of 11 µl Fugene HD, 1 µg pCDNAΔ19 spike-HA, 1 µg p8.91 human immunodeficiency virus 1 (HIV-1) gag-pol expression vector and 1.5 µg pCSFLW (expressing the firefly luciferase reporter gene with the HIV-1 packaging signal). Viral supernatant was collected at 48 and 72 h after transfection, filtered through a 0.45-µm filter and stored at −80 °C as previously described. Infectivity was measured by luciferase detection in target 293T cells transfected with TMPRSS2 and ACE2, Vero E6 ACE2/TMPRSS2, Calu-3, A549 ACE2/TMPRSS2, H1299 and HeLa-ACE2 cells.

Standardization of virus input by SYBR Green-based
product-enhanced PCR assay. The reverse transcriptase (RT) activity of virus preparations was determined by quantitative PCR (qPCR) using a SYBR Green-based product-enhanced PCR assay as previously described 28 . In brief, tenfold dilutions of virus supernatant were lysed in a 1:1 ratio in a 2× lysis solution (made up of 40% glycerol (v/v), 0.25% Triton X-100 (v/v), 100 mM KCl, RNase inhibitor 0.8 U ml −1 , Tris HCl A 12-µl volume of each sample lysate was added to 13 µl of a SYBR Green master mix (containing 0.5 µM MS2-RNA forward and reverse primers, 3.5 pmol ml −1 MS2-RNA and 0.125 U µl −1 Ribolock RNAse inhibitor) and cycled in a QuantStudio. Relative amounts of RT activity were determined as the rate of transcription of bacteriophage MS2 RNA, with absolute RT activity calculated by comparing the relative amounts of RT activity with an RT standard of known activity. comparison between B.1.617.1 and B.1.617.2 Cell culture. Vero E6 TMPRSS2 cells (an African green monkey (Chlorocebus sabaeus) kidney cell line; JCRB1819) 29 were maintained in DMEM (low glucose) (Wako, catalogue no. 041-29775) containing 10% FCS, G418 (1 mg ml −1 ; Nacalai Tesque, catalogue no. G8168-10ML) and 1% antibiotics (penicillin and streptomycin (P/S)).

Viral isolate
Calu-3 cells (a human lung epithelial cell line; ATCC HTB-55) were maintained in minimum essential medium Eagle (Sigma-Aldrich, catalogue no. M4655-500ML) containing 10% FCS and 1% PS. CoV-2 B.1.617.1 versus B.1.617.2  After incubation at 37 °C for 15 min, a maintenance medium (Eagle's minimum essential medium (FUJIFILM Wako Pure Chemical Corporation, catalogue no. 056-08385) including 2% FCS and 1% PS) was added, and the cells were cultured at 37 °C under 5% CO 2 . The cytopathic effect (CPE) was confirmed under an inverted microscope (Nikon), and the viral load of the culture supernatant in which CPE was observed was confirmed by real-time PCR with reverse transcription (RT-PCR). To determine viral genome sequences, RNA was extracted from the culture supernatant using the QIAamp viral RNA mini kit (Qiagen, catalogue no. 52906). A cDNA library was prepared using NEB Next Ultra RNA Library Prep Kit for Illumina (New England Biolab, catalogue no. E7530), and whole-genome sequencing was performed using a Miseq instrument (Illumina).

SARS-
To prepare the working virus, 100 µl of the seed virus was inoculated into Vero E6 TMPRSS2 cells (5,000,000 cells in a T-75 flask). At 1 h after infection, the culture medium was replaced with DMEM (low glucose) (Wako, catalogue no. 041-29775) containing 2% FBS and 1% PS; at 2-3 days post-infection, the culture medium was collected and centrifuged, and the supernatants were collected as the working virus.
The titre of the prepared working virus was measured as 50% tissue culture infectious dose (TCID 50 ). Briefly, 1 day before infection, Vero E6 TMPRSS2 cells (10,000 cells per well) were seeded into a 96-well plate. Serially diluted virus stocks were inoculated onto the cells and incubated at 37 °C for 3 days. The cells were observed by microscopy to judge the CPE appearance. The TCID 50 ml −1 value was calculated with the Reed-Muench method 30 .
One day before infection, 20,000 Calu-3 cells were seeded into a 96-well plate. SARS-CoV-2 (200 TCID 50 ) was inoculated and incubated at 37 °C for 1 h. The infected cells were washed, and 180 µl of culture medium was added. The culture supernatant (10 µl) was collected at indicated time points and used for real-time RT-PCR to quantify the viral RNA copy number.

Real-time RT-PCR.
Real-time RT-PCR was performed as previously described 31,32 . In brief, 5 µl of culture supernatant was mixed with 5 µl of 2× RNA lysis buffer (2% Triton X-100, 50 mM KCl, 100 mM Tris HCl (pH 7.4), 40% glycerol, 0.8 U µl −1 recombinant RNase inhibitor (Takara, catalogue no. 2313B)) and incubated at room temperature for 10 min. RNase-free water (90 µl) was added, and the diluted sample (2.5 µl) was used as the template for real-time RT-PCR performed according to the manufacturer's protocol using the One Step TB Green PrimeScript PLUS RT-PCR kit (Takara, catalogue no. RR096A) and the following primers: forward N, 5′-AGCCTCTTCTCGTTCCTCATC AC-3′; and reverse N, 5′-CCGCCATTGCCAGCCATT C-3′. The copy number of viral RNA was standardized with a SARS-CoV-2 direct detection RT-qPCR kit (Takara, catalogue no. RC300A). The fluorescent signal was acquired using a QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific), a CFX Connect Real-Time PCR Detection System (Bio-Rad) or a 7500 Real Time PCR System (Applied Biosystems).
Virus growth kinetics in HAE cells. Primary nasal HAE cells at the airliquid interface were purchased from Epithelix and the basal MucilAir medium (Epithelix) was changed every 2-3 days for maintenance of HAE cells. All dilution of viruses, wash steps and collection steps were carried out with OptiPRO serum-free medium (SFM; Life Technologies) containing 2× GlutaMAX (Gibco). All wash and collection steps were performed by addition of 200 µl SFM to the apical surface and incubation for 10 min at 37 °C before removing SFM. To infect cells, the basal medium was replaced, and the apical surface of the HAE cells was washed once with SFM to remove mucus before addition of virus to triplicate wells. Cells were infected at a multiplicity of infection (MOI) of 1 × 10 4 genome copies of virus per cell based on E gene qRT-PCR. The cells were incubated with the inoculum for 1 h at 37 °C before washing their apical surface twice and retaining the second wash as the sample for 0 hpi. A single apical wash was performed to collect virus at 24 but not yet available on GISAID).

Titration of outputs from HAE infections.
For quantifying genome copies in the virus inputs and in the supernatant collected from HAE cells, RNA was extracted using the QIAsymphony DSP Virus/Pathogen Mini Kit on the QIAsymphony instrument (Qiagen). qRT-PCR was then performed using the AgPath RT-PCR (Life Technologies) kit on a QuantStudio(TM) 7 Flex System with the primers for SARS-CoV-2 E gene used previously 33 . A standard curve was also generated using dilutions of viral RNA of known copy number to allow quantification of E gene copies in the samples from Ct values. E gene copies per millilitre of original virus supernatant were then calculated.
For measuring infectious virus in samples collected from HAE cells, plaque assays were carried out by performing serial log dilutions of supernatant in DMEM, 1% NEAA and 1% P/S and inoculating onto PBS-washed Vero cells, incubating for 1 h at 37 °C, removing inoculum and overlaying with 1× MEM, 0.2% (w/v) BSA, 0.16% (w/v) NaHCO 3 , 10 mM HEPES, 2 mM l-glutamine, 1× P/S, 0.6% (w/v) agarose. Plates were incubated for 3 days at 37 °C before the overlay was removed and cells were stained for 1 h at room temperature in crystal violet solution.

Lung organoid infection by replication-competent SARS-CoV-2 isolates.
Airway epithelial organoids were prepared as previously reported 10 . For viral infection, primary organoids were passaged and incubated with SARS-CoV-2 in suspension at an MOI of 1 for 2 h. Subsequently, the infected organoids were washed twice with PBS to remove the viral particles. Washed organoids were plated in 20-µl Matrigel domes, cultured in organoid medium and collected at different time points.
Cells were lysed 24 and 48 h post-infection and total RNA was isolated. cDNA was synthesized and qPCR was used to quantify copies of the nucleoprotein gene in samples. A standard curve was prepared using SARS-CoV-2 Positive Control plasmid encoding full nucleocapsid protein (N gene; NEB) and used to quantify copies of the N gene in organoid samples. 18S ribosomal RNA was used as a housekeeping gene to normalize sample-to-sample variation.

Article
Western blotting. Cells were lysed and supernatants were collected 48 h post transfection. Purified virions were prepared by collecting supernatants and passing them through a 0.45-µm filter. Clarified supernatants were then loaded onto a thin layer of 8.4% OptiPrep density gradient medium (Sigma-Aldrich) and placed in a TLA55 rotor (Beckman Coulter) for ultracentrifugation for 2 h at 20,000 r.p.m. The pellet was then resuspended for western blotting. Cells were lysed with cell lysis buffer (Cell Signaling), treated with Benzonase nuclease (70664 Millipore) and boiled for 5 min. Samples were then run on 4-12% Bis Tris gels and transferred onto nitrocellulose or polyvinylidene fluoride membranes using an iBlot or semidry system (Life Technologies and Bio-Rad, respectively).
Membranes were blocked for 1 h in 5% non-fat milk in PBS + 0.1% Tween-20 (PBST) at room temperature with agitation, incubated in primary antibody (anti-SARS-CoV-2 spike, which detects the S2 subunit of SARS-CoV-2 S (Invitrogen, PA1-41165), anti-GAPDH (Proteintech) or anti-p24 (NIBSC)) diluted in 5% non-fat milk in PBST for 2 h at 4 °C with agitation, washed four times in PBST for 5 min at room temperature with agitation and incubated in secondary antibodies anti-rabbit HRP (1:10,000, Invitrogen 31462)and anti-β-actin HRP (1:5,000; sc-47778) diluted in 5% non-fat milk in PBST for 1 h with agitation at room temperature. Membranes were washed four times in PBST for 5 min at room temperature and imaged directly using a ChemiDoc MP imaging system (Bio-Rad).

Virus infection for virion western blotting.
Vero E6 ACE2/TMPRSS2 cells were infected with an MOI of 1 and incubated for 48 h. Supernatant was cleared by a 5-min spin at 300g and then precipitated with 10% PEG6000 (4 h at room temperature). Pellets were resuspended directly in Laemmli buffer with 1 mM dithiothreitol, treated with Benzonase nuclease (70664 Millipore) and sonicated before loading for gel electrophoresis Serum pseudotype neutralization assay Spike pseudotype assays have been shown to have similar characteristics to neutralization testing using fully infectious WT SARS-CoV-2 ( 34 ). Virus neutralization assays were performed on 293T cells transiently transfected with ACE2 and TMPRSS2 using SARS-CoV-2 spike PV expressing luciferase 35 . PV was incubated with serial dilutions of heat-inactivated human serum samples or convalescent plasma in duplicate for 1 h at 37 °C. Virus-and cell-only controls were also included. Then, freshly trypsinized 293T ACE2/TMPRSS2-expressing cells were added to each well. Following a 48-h incubation in a 5% CO 2 environment at 37 °C, the luminescence was measured using the Steady-Glo Luciferase assay system (Promega).
Neutralization assays for convalescent plasma. Convalescent serum samples from HCWs at St Mary's Hospital at least 21 days since PCR-confirmed SARS-CoV-2 infection were collected in May 2020 as part of the REACT2 study.
Convalescent human serum samples were inactivated at 56 °C for 30 min, and replicate serial twofold dilutions (n = 12) were mixed with an equal volume of SARS-CoV-2 (100 TCID 50 ; total volume 100 µl) at 37 °C for 1 h. Vero E6 ACE2/TMPRSS2 cells were subsequently infected with serial fold dilutions of each sample for 3 days at 37 °C. Virus neutralization was quantified via crystal violet staining and scoring for CPE. Each run included 1:5 dilutions of each test sample in the absence of virus to ensure virus-induced CPE in each titration. Back titrations of SARS-CoV-2 infectivity were performed to demonstrate infection with ~100 TCID 50 in each well.

Vaccinee serum neutralization, live virus assays
Vero E6 ACE2/TMPRSS2 cells were seeded at a cell density of 2 × 10 4 per well in a 96-well plate 24 h before infection. Serum was titrated starting at a final 1:10 dilution, with WT (SARS-CoV-2/human/Liverpool/ REMRQ0001/2020), B.1.1.7 or B.1.617.2 virus isolates being added at an MOI of 0.01. The mixture was incubated for 1 h before adding to cells. The plates were fixed with 8% PFA 72 h post-infection and stained with Coomassie blue for 20 min. The plates were washed in water and dried for 2 h. 1% SDS solution was then added to wells and the staining intensity was measured using FLUOstar Omega (BMG Labtech). The percentage of cell survival was determined by comparing the intensity of staining with that in an uninfected well. A nonlinear sigmoidal 4PL model (Graphpad Prism 9.1.2) was used to determine the ID 50 for each serum.

Vesicular stomatitis virus pseudovirus generation for monoclonal antibody assays
Replication-defective vesicular stomatitis virus (VSV) pseudovirus expressing SARS-CoV-2 spike proteins corresponding to the different variants of concern were generated as previously described with some modifications 36 . Lenti-X 293T cells (Takara, 632180) were seeded in 10-cm 2 dishes at a density of 5 × 10 6 cells per dish and the following day transfected with 10 µg of WT or B.1.617.2 spike expression plasmid with TransIT-Lenti (Mirus, 6600) according to the manufacturer's instructions. One day post-transfection, cells were infected with VSV-luc (VSV G) with an MOI of 3 for 1 h, rinsed three times with PBS containing Ca 2+ / Mg 2+ , and then incubated for an additional 24 h in complete medium at 37 °C. The cell supernatant was clarified by centrifugation, filtered (0.45 µm), aliquoted and frozen at −80 °C.
PV neutralization assay for monoclonal antibody. Vero E6 cells expressing TMPRSS2 or not were grown in DMEM supplemented with 10% FBS and seeded into white 96-well plates (PerkinElmer, 6005688) at a density of 20 thousand cells per well. The next day, monoclonal antibodies were serially diluted in pre-warmed complete medium, mixed with WT or B.1.617.2 pseudoviruses and incubated for 1 h at 37 °C in round-bottom polypropylene plates. Medium from cells was aspirated and 50 µl of virus-monoclonal antibody complexes was added to cells and then incubated for 1 h at 37 °C. An additional 100 µl of pre-warmed complete medium was then added on top of complexes, and cells were incubated for an additional 16-24 h. Conditions were tested in duplicate wells on each plate and at least six wells per plate contained untreated infected cells (defining the 0% of neutralization, MAX relative light unit (RLU) value) and infected cells in the presence of S2E12 and S2X259 at 25 µg ml −1 each (defining the 100% of neutralization, MIN RLU value). Medium containing virus-monoclonal antibody complexes was then aspirated from cells and 50 µl of a 1:2 dilution of SteadyLite Plus (Perkin Elmer, 6066759) in PBS with Ca 2+ and Mg 2+ was added to cells. Plates were incubated for 15 min at room temperature and then analysed on the Synergy-H1 (Biotek). The average RLU value for untreated infected wells (MAX RLU ave ) was subtracted by the average MIN RLU (MIN RLU ave ) value and used to normalize the percentage of neutralization of individual RLU values of experimental data according to the following formula: (1 -(RLU x - MIN RLU ave )/(MAX RLU ave - MIN RLU ave )) × 100. Data were analysed and visualized with Prism (Version 9.1.0). IC 50 values were calculated from the interpolated value from the log[inhibitor] versus response, using variable slope (four parameters) nonlinear regression with an upper constraint of ≤100, and a lower constraint equal to 0. Each neutralization assay was conducted on two independent experiments (that is, biological replicates), with each biological replicate containing a technical duplicate. IC 50 values across biological replicates are presented as arithmetic mean ± s.d. The loss or gain of neutralization potency across spike variants was calculated by dividing the variant IC 50 by the WT IC 50 within each biological replicate, and then visualized as arithmetic mean ± s.d.
Corresponding author(s): Ravindra K Gupta (rkg20@cam.ac.uk) Last updated by author(s): Aug 13, 2021 Reporting Summary Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see our Editorial Policies and the Editorial Policy Checklist.

Statistics
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Data analysis
NextClade server v0.14.4 and Pangolin v3.0.5 were used to assign lineages to sequences. Pymol Graphics Suite v2.4.0 was used to visualize and annotate 3D protein structures Stata v13 was used for statistical analyses.
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Sequences from SARS-CoV-2 were obtained from GISAID database (https://gisaid.org/) using the filters and search parameters defined in the methods section. Structural models were obtained from the Protein Data Bank (PDB) https://www.rcsb.org/. All fasta consensus sequences files donated by collaborators are freely available from Gisaid (https://gisaid.org) with accession numbers as follows: Hospital 1: