Amino acids 484 and 494 of SARS-CoV-2 spike are hotspots of immune evasion affecting antibody but not ACE2 binding

  1. Marta Alenquer
  2. Filipe Ferreira
  3. Diana Lousa
  4. Mariana Valério
  5. Mónica Medina-Lopes
  6. Marie-Louise Bergman
  7. Juliana Gonçalves
  8. Jocelyne Demengeot
  9. Ricardo B. Leite
  10. Jingtao Lilue
  11. Zemin Ning
  12. Carlos Penha-Gonçalves
  13. Helena Soares
  14. Cláudio M. Soares
  15. Maria João Amorim

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Abstract

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, with the effect of mutation synergy still incompletely understood. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations, and confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and N501Y. Our analysis of synergic mutations provides a landscape for hotspots for immune evasion and for targets for therapies, vaccines and diagnostics.

One-Sentence Summary

Amino acids in SARS-CoV-2 spike protein implicated in immune evasion are biased for binding to neutralizing antibodies but dispensable for binding the host receptor angiotensin-converting enzyme

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    SciScore for 10.1101/2021.04.22.441007: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsConsent: All participants provided informed consent to take part in the study.
    IRB: This study “Fatores de susceptibilidade genética e proteçao imunológica à COVID-19” was approved on the 25th of May 2020 by the Ethics committee of the Centro Hospitalar Lisboa Ocidental, in compliance with the Declaration of Helsinki, and follows international and national guidelines for health data protection.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For analysis of ACE2 expression, cells were stained with a primary antibody against ACE2 (4µg/ml, R&D Systems, catalog no. AF933) followed by a secondary antibody labelled with Alexa Fluor 568 (1:1000; Life Technologies).
    ACE2
    suggested: (LSBio (LifeSpan Cat# LS-C347-1000, RRID:AB_1271963)
    Plates were washed three times as previously and goat anti-human IgG-HRP secondary antibodies (Abcam, ab97215) were added at 1:25,000 and incubated 30 min at room temperature.
    anti-human IgG-HRP
    suggested: None
    To assess the specificity of our assay, an anti-Spike antibody neutralization assay and an RBD competition assay were performed in SARS-CoV-2 S pseudotyped and vesicular stomatitis virus (VSV) G pseudotyped lentiviral particles, in parallel (fig.
    anti-Spike
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and plasmids: Human hepatocellular carcinoma HuH-7 cells (a kind gift from Dr Colin Adrain, Instituto Gulbenkian de Ciência,
    HuH-7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Portugal), Human Embryonic Kidney 293T (provided by Prof Paul Digard, Roslin Institute, UK) and 293ET cells (from Dr Colin Adrain) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, 21969035) supplemented with 10 % fetal bovine serum (FBS, Gibco, 10500064), 1% penicillin/streptomycin solution (Biowest, L0022) and 2mM L- glutamine (ThermoFisher, 25030024), at 37°C and 5% CO2 atmosphere.
    293ET
    suggested: RRID:CVCL_6996)
    Production of 293T cells stably expressing human ACE2 receptor: To produce VSV-G pseudotyped lentiviruses encoding the human ACE2, 293ET cells were transfected with pVSV-G, psPAX2 and pLEX-ACE2 using jetPRIME (Polyplus), according to manufacturer’s instructions.
    293T
    suggested: None
    In brief, HEK 293T and 293T-ACE2 cells were prepared for flow cytometry analysis by detaching from the wells with trypsin, followed by fixation with 4% paraformaldehyde).
    HEK 293T
    suggested: None
    For the RBD competition assay, 293T-ACE2 cells were pre-incubated with three-fold serial dilutions of SARS-CoV-2 spike’s receptor binding domain, starting with a concentration of 400 µg/ml.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Recombinant DNA
    SentencesResources
    MSC was purchased from Thermo Scientific; psPAX2 and pVSV.
    psPAX2
    suggested: RRID:Addgene_12260)
    G were a kind gift from Dr Luís Moita (Instituto Gulbenkian de Ciência, Portugal); pEGFP-N1 was provided by Dr Colin Crump (University of Cambridge, UK); and pCAGGS containing the SARS-Related Coronavirus 2, Wuhan-Hu-1
    pEGFP-N1
    suggested: RRID:Addgene_111933)
    pCAGGS
    suggested: RRID:Addgene_18926)
    The amplified sequence was cloned back into pCAGGS-SARS-CoV-2-S, between AgeI and SacI restriction sites, generating the expression vector pCAGGS-SARS-CoV-2-Strunc.
    pCAGGS-SARS-CoV-2-S
    suggested: None
    pCAGGS-SARS-CoV-2-Strunc
    suggested: None
    Lentiviral reporter plasmid pLEX-GFP was produced by PCR amplifying GFP from pEGFP-N1 (primers in Table 1) and cloning the insert into BamHI–XhoI restriction sites in the multi-cloning site of pLEX.
    pLEX-GFP
    suggested: None
    For lentiviral plasmid pLEX-ACE2 production, human ACE2 coding sequence was amplified from Huh7 cDNA and cloned into pLEX.
    pLEX-ACE2
    suggested: None
    pLEX
    suggested: RRID:Addgene_117987)
    Production of 293T cells stably expressing human ACE2 receptor: To produce VSV-G pseudotyped lentiviruses encoding the human ACE2, 293ET cells were transfected with pVSV-G, psPAX2 and pLEX-ACE2 using jetPRIME (Polyplus), according to manufacturer’s instructions.
    pVSV-G
    suggested: RRID:Addgene_138479)
    Production and titration of spike pseudotyped lentiviral particles: To generate spike pseudotyped lentiviral particles, 3×106 293ET cells were co-transfected with 8.89ug pLex-GFP reporter, 6.67ug psPAX2, and 4.44ug pCAGGS-SARS-CoV-2-S WT or mutants (or pVSV.G, as a control), using jetPRIME according to manufacturer’s instructions.
    pVSV . G
    suggested: None
    Software and Algorithms
    SentencesResources
    MSC was purchased from Thermo Scientific; psPAX2 and pVSV.
    Thermo Scientific
    suggested: (Thermo Scientific Wellwash Wellwash, RRID:SCR_020569)
    G were a kind gift from Dr Luís Moita (Instituto Gulbenkian de Ciência, Portugal); pEGFP-N1 was provided by Dr Colin Crump (University of Cambridge, UK); and pCAGGS containing the SARS-Related Coronavirus 2, Wuhan-Hu-1
    Portugal)
    suggested: None
    Analysis of cell populations was performed in a Becton Dickinson (BD) LSR Fortessa X-20 equipped with FACS Diva and FlowJo (BD, Franklin Lakes, NJ) software’s
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The half maximal inhibitory concentration (IC50) was determined using four-parameter nonlinear regression (least squares regression without weighting; constraints: bottom=0) (GraphPad Prism 9).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    This was done for all PDB structures using in-house Python scripts.
    Python
    suggested: (IPython, RRID:SCR_001658)
    Finally, the numerical python library (NumPy (97)) was used to calculate the frequency of contact for each spike protein residue with an antibody residue, from the sample of PDB structures analyzed.
    NumPy
    suggested: (NumPy, RRID:SCR_008633)
    MD simulation of the RBD-ACE2 complex: Molecular dynamics (MD) simulations of the RBD bound to the ACE2 protein were performed with the GROMACS 2020.3 package (98), using the Amber14sb (99) force field, starting from the 6m0j structure (12), in a truncated dodecahedron box filled with water molecules (minimum of 1.2 nm between protein and box walls).
    GROMACS
    suggested: (GROMACS, RRID:SCR_014565)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Our approach has some caveats. Regarding the neutralization assays, and despite spike being the immunodominant protein targeted by antibodies (7–11), other viral proteins may contribute, even if in a small proportion to neutralization activity in vivo, which we are unable to detect using spike-pseudotyped particles. In addition, the sera/plasma we used was obtained at a fixed time interval. Future experiments should repeat the analysis using sera/plasma of people infected with known genotypes, or from different geographical areas and obtained at different times along the epidemics dynamic. Finally, we inspected a handful of mutations and not a comprehensive mutational map across spike (2, 4). However, we traced several mutations across spike in single and multiple combinations, and thus, we conclude that our analysis is relevant to inform on mutation-driven escape viruses to antibodies. The analysis of frequency of interactions between spike-antibodies or ACE2 has some issues associated as well. The pool of complexes spike protein/RBD and antibodies analyzed here is not necessarily representative of the universe of antibodies found in COVID-19 patients, since it is limited by the availability of structural information and is biased to monoclonal antibodies (table S4). All the antibodies analyzed are neutralizing antibodies, which is not the case in nature (53). Additionally, many structural studies have focused on the interaction with the RBD, while other regions have not bee...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2021.04.22.441007 on bioRxiv