The SARS-CoV-2 Delta variant induces an antibody response largely focused on class 1 and 2 antibody epitopes

  1. Allison J. Greaney
  2. Rachel T. Eguia
  3. Tyler N. Starr
  4. Khadija Khan
  5. Nicholas Franko
  6. Jennifer K. Logue
  7. Sandra M. Lord
  8. Cate Speake
  9. Helen Y. Chu
  10. Alex Sigal
  11. Jesse D. Bloom

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Abstract

Exposure histories to SARS-CoV-2 variants and vaccinations will shape the specificity of antibody responses. To understand the specificity of Delta-elicited antibody immunity, we characterize the polyclonal antibody response elicited by primary or mRNA vaccine-breakthrough Delta infections. Both types of infection elicit a neutralizing antibody response focused heavily on the receptor-binding domain (RBD). We use deep mutational scanning to show that mutations to the RBD’s class 1 and class 2 epitopes, including sites 417, 478, and 484–486 often reduce binding of these Delta-elicited antibodies. The anti-Delta antibody response is more similar to that elicited by early 2020 viruses than the Beta variant, with mutations to the class 1 and 2, but not class 3 epitopes, having the largest effects on polyclonal antibody binding. In addition, mutations to the class 1 epitope (e.g., K417N) tend to have larger effects on antibody binding and neutralization in the Delta spike than in the D614G spike, both for vaccine- and Delta-infection-elicited antibodies. These results help elucidate how the antigenic impacts of SARS-CoV-2 mutations depend on exposure history.

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  1. Version published to 10.1371/journal.ppat.1010592
  2. ScreenIT

    SciScore for 10.1101/2022.03.12.484088: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: In all studies, written informed consent was obtained from each participant.
    IRB: Blood samples were obtained after written informed consent from adults with PCR-confirmed SARS-CoV-2 infection who were enrolled in a prospective cohort study at the Africa Health Research Institute approved by the Biomedical Research Ethics Committee at the University of KwaZulu–Natal (reference BREC/00001275/2020).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For RBD expression experiments, 45 OD units of yeast were labeled in 1:100 diluted chicken-anti-Myc-FITC antibody (Immunology Consultants CMYC45F) to detect the RBD’s C-terminal Myc tag.
    Myc tag .
    suggested: None
    These measurements were used in downstream steps to computationally filter library variants that were highly deleterious for RBD expression or ACE2 binding and would likely represent spurious antibody-escape mutations (see below for details).
    ACE2
    suggested: None
    After the plasma incubations, the libraries were secondarily labeled for 1 hour with 1:100 fluorescein isothiocyanate-conjugated anti-MYC antibody (Immunology Consultants Lab, CYMC-45F) to label for RBD expression and 1:200 Alexa Fluor-647-conjugated goat anti-human-IgA+IgG+IgM (Jackson ImmunoResearch 109-605-064) to label for bound plasma antibodies.
    anti-MYC
    suggested: None
    anti-human-IgA+IgG+IgM
    suggested: None
    , version 0.8.10) to process Illumina sequences into counts of each barcoded RBD variant in each pre-selection and antibody-escape population.
    antibody-escape population .
    suggested: None
    S4B), and are the counts of variant v in the RBD library after and before enriching for antibody-escape variants with a pseudocount of 0.5 added to all counts, and and are the total counts of all variants after and before the antibody-escape enrichment.
    antibody-escape enrichment .
    suggested: None
    Antibody binding was detected with TMB/E HRP substrate (Millipore Sigma, ES001) and 1 N HCl was used to stop the reaction.
    ES001
    suggested: None
    The multidimensional scaling in Fig 4 and 5 that projects the antibody-escape maps into two dimensions was performed using the Python scikit-learn package, version 1.0.1.
    antibody-escape
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To generate spike-pseudotyped lentiviral particles (58), 6 × 105 HEK-293T (ATCC CRL-3216) cells per well were seeded in 6-well plates in 2 mL D10 growth media (Dulbecco’s Modified Eagle Medium with 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin).
    HEK-293T
    suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)
    100 µL of serially diluted spike-pseudotyped lentiviral particles were added to 1.25 × 104 293T-ACE2 cells (BEI Resources NR-52511), grown overnight in 50 µL of D10 growth media in a 96-well black-walled poly-L-lysine coated plate (Greiner Bio-One, 655936)
    293T-ACE2
    suggested: None
    Note that these assays were performed in 293T cells over-expressing human ACE2, which emphasize contributions of ACE2-competitive antibodies to viral neutralization (3,34,60).
    293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    For experiments involving D614G spike, we used spike-pseudotyped lentiviral particles that were generated essentially as described in (58), using a codon-optimized SARS-CoV-2 spike from Wuhan-Hu-1 strain that contains a 21-amino-acid deletion at the end of the cytoplasmic tail (59) and the D614G mutation that is now predominant in human SARS-CoV-2 (25).
    Wuhan-Hu-1
    suggested: None
    Depletion of RBD-binding antibodies from polyclonal sera: Magnetic beads conjugated to the SARS-CoV-2 Wuhan-Hu-1 RBD (ACROBiosystems, MBS-K002) or Delta RBD (ACROBiosystems, MBS-K037) were prepared according to the manufacturer’s protocol.
    SARS-CoV-2 Wuhan-Hu-1 RBD
    suggested: None
    Recombinant DNA
    SentencesResources
    AWY101 yeast containing an empty vector pETcon plasmid were grown overnight, shaking, at 30°C in SG-CAA media.
    pETcon
    suggested: RRID:Addgene_41522)
    Software and Algorithms
    SentencesResources
    PacBio HiFi circular consensus sequences (CCSs) were generated on-instrument (Sequel IIe control software version 10.1.0.119549) and demultiplexed with lima using SMRTLink version 10.1.0.119588.
    SMRTLink
    suggested: None
    The multidimensional scaling in Fig 4 and 5 that projects the antibody-escape maps into two dimensions was performed using the Python scikit-learn package, version 1.0.1.
    Python
    suggested: (IPython, RRID:SCR_001658)
    scikit-learn
    suggested: (scikit-learn, RRID:SCR_002577)

    Results from OddPub: Thank you for sharing your code and data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Our study has several limitations. First, each cohort in this study consists of a relatively small sample size. Due to patterns of SARS-CoV-2 viral circulation and competition among variants (e.g., early 2020, Beta, and Delta viruses circulated during different time periods and in different geographic regions), as well as the introduction of SARS-CoV-2 vaccination in 2021, the cohorts differ with regards to geographic location, age, and time of sample collection. Second, the antibody-escape mapping experiments were performed in a yeast-displayed deep mutational scanning system which only measures antibody binding to the RBD. There is not always a direct relationship between antibody binding and neutralization, as some epitopes are more immunodominant with regards to neutralization than others, and the relative contributions to neutralization of antibodies targeting different spike epitopes may depend on cell type and ACE2 expression level used in the experimental assays (3,34,35). Despite these caveats, our results have important implications for future viral evolution and vaccine design. Due to the rapid antigenic evolution of SARS-CoV-2, public health experts must decide if and when to update SARS-CoV-2 vaccines (51,52). But because each variant elicits an antibody response with its own susceptibilities to erosion by mutation, the impacts of mutations in future variants may depend on prior exposure history. This knowledge will help to understand which vulnerabilities may ex...

    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: 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.

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

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  3. Version published to 10.1101/2022.03.12.484088v1 on bioRxiv