B.1.617.3 SARS CoV-2 spike E156G/Δ157-158 mutations contribute to reduced neutralization sensitivity and increased infectivity

  1. Tarun Mishra
  2. Garima Joshi
  3. Atul Kumar
  4. Rishikesh Dalavi
  5. Pankaj Pandey
  6. Sanjeev Shukla
  7. Ram Kumar Mishra
  8. Ajit Chande

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Abstract

SARS CoV-2 variants raise significant concerns due to their ability to cause vaccine breakthrough infections. Here, we sequence-characterized the spike gene, isolated from a breakthrough infection, that corresponded to B.1.617.3 lineage. Delineating the functional impact of spike mutations using reporter pseudoviruses (PV) revealed that N-terminal domain (NTD)-specific E156G/Δ157-158 contributed to increased infectivity and reduced sensitivity to ChAdOx1 nCoV-19 vaccine (Covishield™)-elicited neutralizing antibodies. A six-nucleotide deletion (467-472) in the spike coding region introduced this change in the NTD. We confirmed the presence of E156G/Δ157-158 in the RT-PCR-positive cases concurrently screened, in addition to other circulating spike (S1) mutations like T19R, T95I, L452R, E484Q, and D614G. Notably, E156G/Δ157-158 was present in more than 85% of the sequences reported from the USA, UK, and India in August 2021. The spike PV bearing combination of E156G/Δ157-158 and L452R further promoted infectivity and conferred immune evasion. Additionally, increased cell-to-cell fusion was observed when spike harbored E156G/Δ157-158, L452R, and E484Q, suggesting a combinatorial effect of these mutations. Notwithstanding, the plasma from a recovered individual robustly inhibited mutant spike PV, indicating the increased breadth of neutralization post-recovery. Our data highlights the importance of spike NTD-specific changes in determining infectivity and immune escape of variants.

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  1. ScreenIT

    SciScore for 10.1101/2021.10.04.463028: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: The Institute Ethics committee approved this study.
    Consent: All the samples were collected with due consent from the donors.
    Sex as a biological variablenot detected.
    RandomizationTo calculate spike mutation’s effect on syncytium formation, five different fields were randomly chosen, and the area of fused cells was measured using ImageJ software.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For the p24 and beta-actin detection anti p24 (NIH ARP), rabbit anti-beta actin (LI-COR Biosciences, Cat# 926-42210, RRID:AB_1850027), for SARS CoV-2 spike detection mouse anti-spike (Cat# ZMS1076
    rabbit anti-beta actin
    detected: (LI-COR Biosciences Cat# 926-42210, RRID:AB_1850027)
    The IR dye 680 goat anti-mouse was used as a secondary antibody for the anti-spike antibody.
    anti-mouse
    suggested: None
    anti-spike
    suggested: None
    The IR dye 800 goat anti-mouse, or IR dye 800 goat anti-rabbit (LI-COR Biosciences Cat# 925-68070, RRID:AB_2651128, and LI-COR Biosciences Cat# 925-32211, RRID:AB_2651127) were used against primary p24 and beta-actin antibody respectively.
    anti-mouse
    detected: (LI-COR Biosciences Cat# 925-68070, RRID:AB_2651128)
    detected: (LI-COR Biosciences Cat# 925-32211, RRID:AB_2651127)
    beta-actin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    For infectivity experiments, HEK293T ACE2+ cells were seeded 24h before transduction.
    HEK293T ACE2+
    suggested: None
    Separately, HEK293T cells were co-transfected with pEGFPN1 (50ng) and ACE2 expressing vector (500ng).
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    psPAX2 (6μg)
    psPAX2
    suggested: RRID:Addgene_12260)
    Separately, HEK293T cells were co-transfected with pEGFPN1 (50ng) and ACE2 expressing vector (500ng).
    pEGFPN1
    suggested: RRID:Addgene_109293)
    Software and Algorithms
    SentencesResources
    To calculate spike mutation’s effect on syncytium formation, five different fields were randomly chosen, and the area of fused cells was measured using ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Software and Statistical Analysis: All graphs were generated using GraphPad Prism (version 9.0).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Pymol (version 1.2) and Alpha-fold were used for protein structure visualization.
    Pymol
    suggested: (PyMOL, RRID:SCR_000305)
    Image-J was used for image processing.
    Image-J
    suggested: (ImageJ, RRID:SCR_003070)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    One of the limitations was that we did not have access to the plasma from before infection to appreciate the vaccine efficacy for the subject case. Furthermore, undoubtedly, the sample size would have further strengthened the study. However, we could not have had more samples that were matched for geographical location, the time intervals between the two doses (later on changed) given the limited numbers of vaccinated individuals then. Within these limitations, our results have important implications suggesting the possibility that NTD specific changes, like the ones reported here, might be driving the spread of variants. We have confirmed that the L452R mutation in the RBD, prevalent in the Delta, Lambda, and epsilon variants, contributes to enhancing infectivity 4,21,30. The Delta and Lambda variants under VOI and VOC, respectively, do not alone rely on the higher infectivity for spread but also showed immune evasion from the vaccine-induced antibodies 7,14,20. In contrast, the Epsilon variant is now removed from the VOC/VOI list because of its low spreading capacity, although being more infectious than the spike bearing D614G. This suggests that higher infectivity alone is not sufficient for the spread. As shown here, the combined effect of E156G/Δ157-158 and L452R mutations that maintain higher infectivity and reduced neutralization susceptibility may underlie a particular variant’s dominance. The growing evidence that NTD-specific mutations are modulating antigenicity re...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 23. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.10.04.463028v1 on bioRxiv