key: cord-0937822-wp18z6rm
authors: Ferreira, Isabella; Kemp, Steven; Datir, Rawlings; Saito, Akatsuki; Meng, Bo; Rakshit, Partha; Takaori-Kondo, Akifumi; Kosugi, Yusuke; Uriu, Keiya; Kimura, Izumi; Shirakawa, Kotaro; Abdullahi, Adam; Agarwal, Anurag; Ozono, Seiya; Tokunaga, Kenzo; Sato, Kei; Gupta, Ravindra K
title: SARS-CoV-2 B.1.617 mutations L452 and E484Q are not synergistic for antibody evasion
date: 2021-07-14
journal: J Infect Dis
DOI: 10.1093/infdis/jiab368
sha: 6797e13a12b37beb10f79a33ac324fb76b02ba47
doc_id: 937822
cord_uid: wp18z6rm

The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that two key mutations seen in the receptor binding domain L452R and E484Q would have additive effects on evasion of neutralising antibodies. We report that spike bearing L452R and E484Q confers modestly reduced sensitivity to BNT162b2 mRNA vaccine-elicited antibodies following either first or second dose. The effect is similar in magnitude to the loss of sensitivity conferred by L452R or E484Q alone. These data demonstrate reduced sensitivity to vaccine elicited neutralising antibodies by L452R and E484Q but lack of synergistic loss of sensitivity.

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The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that two key mutations seen in the receptor binding domain L452R and E484Q would have additive effects on evasion of neutralising antibodies. We report that spike bearing L452R and E484Q confers modestly reduced sensitivity to BNT162b2 mRNA vaccine-elicited antibodies following either first or second dose. The effect is similar in magnitude to the loss of sensitivity conferred by L452R or E484Q alone. These data demonstrate reduced sensitivity to vaccine elicited neutralising antibodies by L452R and E484Q but lack of synergistic loss of sensitivity. India experienced a wave of infections in mid 2020 that was controlled by a nationwide lockdown.

Since easing of restrictions, India has seen expansion in cases of COVID-19 since March 2021. The B.1.617 variant emerged in the state of Maharashtra in late 2020/early 2021 and has spread throughout India and to at least 60 countries. It was labelled initially as a 'double mutant' since two of the mutations L452R and E484Q were matched to an in-house screening database for mutations leading to probable evasion of antibodies and/or being linked to increased transmissibility.

L452R and E484Q are located in the critical receptor binding domain that interacts with ACE2 4 . L452R was observed in the 'Epsilon Variant' B.1.429 and is associated with increase in viral load and around 20% increased transmissibility 5 . It was also associated with increased ACE2 binding, increased infectivity 6 and 3-6 fold loss of neutralisation sensitivity to vaccine elicited sera in experiments with pseudotyped virus (PV) particles 6, 7 . Little is known about E484Q, though E484K is a defining feature of two VOCs, B.1.351 and P.1, and is found alongside K417N/T as well as N501Y in these VOC. E484K has also emerged in the background of B.1.1.7 8 

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All sequences excluding low-quality sequences (>5% N regions) with the L452R mutation were downloaded from https://gisaid.org on the 4 th May 2021 and manually aligned to reference strain MN908947.3 with mafft v4.475 using the --keeplength --addfragments option. Sequences were deduplicated using bbtools dedupe.sh. A random subset of 400 global sequences (excluding USA), and 100 USA sequences were then selected with seqtk and concatenated. Sequence lineages were assigned to all sequences with pangolin v2.4 (https://github.com/cov-lineages/pangolin) and pangolearn (04/05/2021).

Phylogenies were then inferred using maximum-likelihood in IQTREE v2.1.3 9 using a GTR+R6 model and the -fast option. Mutations of interest were determined using a local instance of nextclade-cli v0.14.2 (https://github.com/nextstrain/nextclade). The inferred phylogeny was annotated in R v4.04 using ggtree v2.2.4 and rooted on the SARS-CoV-2 reference sequence, and nodes arranged in descending order. Major lineages were annotated on the phylogeny, as well as a heatmap indicating which mutations of interest were carried by each viral sequence.

The PyMOL Molecular Graphics System v. 

The reverse transcriptase activity of virus preparations was determined by qPCR using a SYBR Greenbased product-enhanced PCR assay (SG-PERT) as previously described 11 . Briefly, 10-fold dilutions of virus supernatant were lysed in a 1:1 ratio in a 2x lysis solution (made up of 40% glycerol v/v 0.25% Trition X-100 v/v 100mM KCl, RNase inhibitor 0.8 U/ml, TrisHCL 100mM, buffered to pH7.4) for 10 minutes at room temperature.

Virus neutralisation assays were performed on 293T cell transiently transfected with ACE2 and TMPRSS2 using SARS-CoV-2 spike pseudotyped virus expressing luciferase 12 . Pseudotyped virus was incubated with serial dilution of heat inactivated human serum samples or convalescent plasma in duplicate for 1h 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 48h incubation in a 5% CO 2 environment at 37°C, the luminescence was measured using Steady-Glo Luciferase assay system (Promega). IC50 was calculated in GraphPad Prism v8.0.

The ACE2-expressing lentiviral plasmid pWPI-ACE2-zeo was generated by replacing the original EGFP Thermo Ficher) and G418 (400 μg ml −1 ; Nacalai) selections for 14 d.

We subsampled SARS-CoV-2 sequences containing L452R from GISAID, and inferred a maximum likelihood phylogenetic tree ( Figure 1A) . We annotated the sequences based on the accompanying Spike mutations L452R and E484Q are in the receptor binding domain that not only binds ACE2 13 , but is a target for neutralising antibodies 14, 15 (Figure 2A) . We tested the neutralisation sensitivity of A c c e p t e d M a n u s c r i p t 10 combinations of mutations found in B.1.617.1: L452R, E484Q and P681R, using a previously reported pseudotyped virus (PV) system. We tested 24 stored sera from first dose ( Figure 2B ) and 16 sera from second dose ( Figure 2C ) Pfizer BNT162b2 vaccinees against a range of spike mutation bearing PV ( Figure 2B,C, Supplementary Figure 1B,C) . E484Q had a similar impact on reducing neutralisation sensitivity as L452R and E484K (3.6-4.5 fold). When E484Q and L452R were combined, there was a statistically significant loss of sensitivity as compared to wild type, but the fold change of 5.1 was similar to that observed with each mutation individually with absence of evidence for an additive effect ( Figure 2B, Supplementary Figure 1B) . However, as expected, in some sera there was evidence for variable neutralising activity against the L452R and E484Q PVs, reflecting differential antibody responses between individuals. When we tested second dose sera ( Figure 2C , Supplementary Figure   1C ), similar patterns were observed between different viruses although fold changes were lower overall, likely due to increased neutralisation breadth and potency following booster vaccination 8 .

Finally, with the PV system we measured spike mediated entry into target HOS cells endogenously expressing ACE2 and TMPRSS2 receptors. The E484K and L452R mutant did not have significantly higher entry efficiency compared to single mutants ( Figure 2D ). We also tested the entry efficiency of L452R, E484Q and P681R in a range of target cell lines, either exogenously or endogenously expressing SARS-CoV-2 receptors ACE2/TMPRSS2. The spike triple mutant exhibited similar or mildly reduced entry compared to Wuhan-1 D614G spike (Supplementary Figure 2) .

Here we demonstrate three lineages of B.1.617, all bearing the L452R mutation. We report key differences in amino acids between sub-lineages and focus on B.1.617.1 bearing two key RBD A c c e p t e d M a n u s c r i p t 11 mutations L452R, E484Q. In vitro, we find modestly reduced sensitivity of the spike protein bearing RBD mutations L452R and E484Q to BNT162b2 mRNA vaccine-elicited antibodies that is similar in magnitude to the loss of sensitivity conferred by L452R or E484Q alone. P681R did not appear to alter the sensitivity to vaccine sera or to alter the entry efficiency conferred by spike protein on lentiviral particles. These data demonstrate reduced sensitivity to vaccine elicited neutralising antibodies by the RBD bearing L452R and E484Q but lack of synergistic loss of sensitivity. M a n u s c r i p t 

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We would like to thank the Department of Biotechnology, NCDC, RKG is supported by a Wellcome 

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