SARS-CoV-2 B.1.617 Mutations L452R and E484Q Are Not Synergistic for Antibody Evasion

Abstract The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that 2 key mutations seen in the receptor-binding domain, L452R and E484Q, would have additive effects on evasion of neutralizing 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 neutralizing antibodies by L452R and E484Q but lack of synergistic loss of sensitivity.

The SARS-CoV-2 B.1.617 variant emerged in the Indian state of Maharashtra in late 2020. There have been fears that 2 key mutations seen in the receptor-binding domain, L452R and E484Q, would have additive effects on evasion of neutralizing 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 neutralizing antibodies by L452R and E484Q but lack of synergistic loss of sensitivity.
Global control of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has yet to be realized despite availability of highly effective vaccines. Emergence of new variants with multiple mutations is likely the result of chronic infections within individuals who are immune compromised [1]. These new variants with antibody escape mutations have coincided with vaccine scale up, potentially threatening their success in controlling the pandemic.
India experienced a wave of infections in mid-2020 that was controlled by a nationwide lockdown. After easing of restrictions, India has seen expansion in cases of coronavirus disease 2019 (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 because 2 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 (RBD) that interacts with angiotensin-converting enzyme 2 (ACE2) [2]. L452R was observed in the epsilon variant B.1.429 and is associated with increase in viral load and around 20% increased transmissibility [3]. It was also associated with increased ACE2 binding, increased infectivity [4], and 3-to 6-fold loss of neutralization sensitivity to vaccine-elicited sera in experiments with pseudotyped virus (PV) particles [5]. Little is known about E484Q, although E484K is a defining feature of 2 variants of concern (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 [6].
Phylogenies were then inferred using maximum-likelihood in IQTREE version 2.1.3 [7] using a GTR + R6 model and the -fast option. Mutations of interest were determined using a local instance of nextclade-cli version 0.14.2 (https://github. com/nextstrain/nextclade). The inferred phylogeny was annotated in R version 4.04 using ggtree version 2.2.4 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.

Structural Analyses
The PyMOL Molecular Graphics System version 2.4.0 (https:// github.com/schrodinger/pymol-open-source/releases) was used to map the location of the 2 RBD mutants L452R and E484Q onto 2 previously published SARS-CoV-2 spike glycoprotein structures. The 2 structures included a closedconformation spike protein, Protein Data Bank 6ZGE and a spike protein in open conformation, bound to nAb H4 [8].

Serum Samples and Ethical Approval
Ethical approval was sought for use of serum samples. Controls with COVID-19 were enrolled to the National Institute of Health Research BioResource Centre Cambridge under ethics review board (17/EE/0025). Protocols involving human subjects recruited at Kyoto University, Japan, were reviewed and approved by (approval numbers G0697 and G1309). All human subjects provided written informed consent.

Cells
HEK 293T CRL-3216, Vero CCL-81 were purchased from American Type Culture Collection and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 mg/ mL streptomycin. All cells were regularly tested and were mycoplasma free.

Pseudotype Virus Preparation
Plasmids encoding the spike protein of SARS-CoV-2 with a C terminal 19 amino acid deletion with D614G were used. Mutations were introduced using Quickchange Lightning Site-Directed Mutagenesis kit (Agilent) following the manufacturer's instructions. 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 of Fugene HD, 1 µg of pCDNAΔ19 spike-HA, 1 µg of p8.91 HIV-1 gag-pol expression vector, and 1.5 µg of pCSFLW (expressing the firefly luciferase reporter gene with the HIV-1 packaging signal). Viral supernatant was collected at 48 and 72 hours after transfection, filtered through a 0.45-µm filter and stored at −80°C. Infectivity was measured by luciferase detection in target 293T cells transfected with TMPRSS2 and ACE2.

Serum Pseudotype Neutralization Assay for Pfizer BNT162b2 Dose 1 Experiments
Virus neutralization assays were performed on 293T cells transiently transfected with ACE2 and TMPRSS2 using SARS-CoV-2 spike PV expressing luciferase [10]. PV was incubated with serial dilution of heat inactivated human serum samples in duplicate for 1h at 37°C. Virus and cell-only controls were also included. Then, freshly trypsinized 293T ACE2/TMPRSS2expressing cells were added to each well. Following 48-hour incubation in a 5% CO 2 environment at 37°C, the luminescence was measured using Steady-Glo Luciferase assay system (Promega). Half maximum inhibitory concentration (IC 50 ) was calculated in GraphPad Prism version 8.0.

Establishment of Stable Cells for Pfizer Dose 2 Experiments
The ACE2-expressing lentiviral plasmid pWPI-ACE2-zeo was generated by replacing the original EGFP gene of the lentiviral transfer plasmid pWPI [11], with the zeocin-resistant gene, and by inserting the ACE2 gene into the region immediately upstream of the internal ribosome entry site. Similarly, the TMPRSS2-expressing lentiviral plasmid pWPI-TMPRSS2-neo was created by inserting the neomycin-resistant gene and the TMPRSS2 gene into pWPI.

RESULTS
We subsampled SARS-CoV-2 sequences containing L452R from GISAID database, and inferred a maximum likelihood phylogenetic tree ( Figure 1A). We annotated the sequences based on the accompanying mutations and observed 3 lineages within B.  increasing in India ( Figure 1B), although with the caveat of very low sequencing of prevalent cases and heterogeneous sampling across the country. Spike mutations L452R and E484Q are in the RBD that not only binds ACE2 [13] but is also a target for neutralizing antibodies [14,15] (Figure 2A). We tested the neutralization sensitivity of combinations of mutations found in B.1.617.1 (L452R, E484Q, and P681R) using a previously reported 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 and 2C and Supplementary Figure 1B and 1C). E484Q had a similar impact on reducing neutralization 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 Figure 1B). However, as expected, in some sera there was evidence for variable neutralizing activity against the L452R and E484Q PVs, reflecting differential antibody responses between individuals. When we tested second-dose sera ( Figure 2C and Supplementary Figure 1C), similar patterns were observed between different viruses although fold changes were lower overall, likely due to increased neutralization 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).

DISCUSSION
Here we demonstrate 3 lineages of B.1.617, all bearing the L452R mutation. We report key differences in amino acids between sublineages and focus on B.1.617.1 bearing 2 key RBD 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 neutralizing antibodies by the RBD bearing L452R and E484Q but lack of synergistic loss of sensitivity.

Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.