A SARS-CoV-2 variant elicits an antibody response with a shifted immunodominance hierarchy

  1. Allison J. Greaney
  2. Tyler N. Starr
  3. Rachel T. Eguia
  4. Andrea N. Loes
  5. Khadija Khan
  6. Farina Karim
  7. Sandile Cele
  8. John E. Bowen
  9. Jennifer K. Logue
  10. Davide Corti
  11. David Veesler
  12. Helen Y. Chu
  13. Alex Sigal
  14. Jesse D. Bloom

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Abstract

Many SARS-CoV-2 variants have mutations at key sites targeted by antibodies. However, it is unknown if antibodies elicited by infection with these variants target the same or different regions of the viral spike as antibodies elicited by earlier viral isolates. Here we compare the specificities of polyclonal antibodies produced by humans infected with early 2020 isolates versus the B.1.351 variant of concern (also known as Beta or 20H/501Y.V2), which contains mutations in multiple key spike epitopes. The serum neutralizing activity of antibodies elicited by infection with both early 2020 viruses and B.1.351 is heavily focused on the spike receptor-binding domain (RBD). However, within the RBD, B.1.351-elicited antibodies are more focused on the “class 3” epitope spanning sites 443 to 452, and neutralization by these antibodies is notably less affected by mutations at residue 484. Our results show that SARS-CoV-2 variants can elicit polyclonal antibodies with different immunodominance hierarchies.

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  1. Version published to 10.1371/journal.ppat.1010248
  2. ScreenIT

    SciScore for 10.1101/2021.10.12.464114: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Description of cohort and ethics statement: Samples were collected from participants enrolled in a prospective cohort study approved by the Biomedical Research Ethics Committee (BREC) at the University of KwaZulu–Natal (reference BREC/00001275/2020).
    Consent: Written informed consent was obtained from each participant.
    Sex as a biological variableFour were males and 5 were females.
    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
    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
    Magnetic Separation Rack, Thermo Fisher Scientific, CS15000) was used to separate antibodies that bind RBD from the supernatant, and the supernatant (the post-RBD antibody depletion sample) was removed.
    post-RBD
    suggested: None
    Note that these assays were performed in 293T cells over-expressing human ACE2, which may underestimate contributions of non-RBD-binding antibodies to viral neutralization (7, 35, 60).
    ACE2
    suggested: None
    Dilution series of the synthetic sera comprised of the anti-RBD antibody REGN10987 (72), which binds to both Wuhan-1-like RBD and B.1.351 RBD, and pooled pre-pandemic human serum from 2017-2018 (Gemini Biosciences; nos. 100–110, lot H86W03J; pooled from 75 donors) were performed such that the anti-spike antibody was present at a highest concentration of 0.25 µg/mL.
    anti-spike
    suggested: None
    Pre-pandemic serum alone, without anti-RBD antibody depletion, was used as a negative control, averaged over 2 replicates
    anti-RBD
    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
    Experimental Models: Cell Lines
    SentencesResources
    Generation of pseudotyped lentiviral particles: HEK-293T (American Type Culture Collection, CRL-3216) cells were used to generate SARS-CoV-2 spike-pseudotyped lentiviral particles and 293T-ACE2 cells (Biodefense and Emerging Infectious Research Resources Repository (BEI Resources), NR-52511) were used to titer the SARS-CoV-2 spike-pseudotyped lentiviral particles and to perform neutralization assays (see below).
    293T-ACE2
    suggested: None
    To generate spike-pseudotyped lentiviral particles (70), 6e105 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)
    Note that these assays were performed in 293T cells over-expressing human ACE2, which may underestimate contributions of non-RBD-binding antibodies to viral neutralization (7, 35, 60).
    293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Experimental Models: Organisms/Strains
    SentencesResources
    For experiments involving D614G spike, we used spike-pseudotyped lentiviral particles that were generated essentially as described in (70), 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 (27) and the D614G mutation that is now predominant in human SARS-CoV-2 (30).
    Wuhan-Hu-1
    suggested: None
    Recombinant DNA
    SentencesResources
    AWY101 yeast containing a negative control (containing an empty vector pETcon plasmid) were grown overnight at 30°C in galactose-containing media.
    pETcon
    suggested: RRID:Addgene_41522)
    Software and Algorithms
    SentencesResources
    The resulting CCSs are available on the NCBI Sequence Read Archive, BioProject PRJNA770094,
    NCBI Sequence Read Archive
    suggested: (NCBI Sequence Read Archive (SRA, RRID:SCR_004891)
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)
    Statistical Analysis: The percent of neutralizing activity of early-2020 and B.1.351-convalescent plasmas due to RBD-binding antibodies is plotted with the plotnine python package, version 0.8.0 (https://plotnine.readthedocs.io/en/stable/index.html), shown as a Tukey boxplot (middle line indicating median, box limits indicating interquartile range) with individual measurements overlaid as points.
    python
    suggested: (IPython, RRID:SCR_001658)
    All raw sequencing data are available on the NCBI Short Read Archive at BioProject PRJNA770094, BioSample SAMN22208699, SAMN22208700.
    NCBI Short Read Archive
    suggested: None

    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. The cohorts of individuals infected with early 2020 and B.1.351 viruses are small, and are geographically and temporally distinct. Nevertheless, the two cohorts are relatively well-matched with respect to age, sex, and days-post symptom onset of sample collection (Table 1) and assays were performed under comparable conditions. Our deep mutational scanning measured binding to yeast-displayed RBD, which may not capture all relevant features of full-length spike in the context of virus. Finally, our neutralization assays used pseudotyped lentiviral particles and ACE2-overexpressing cells, and some recent works suggest that the relative importance of different spike epitopes for neutralization can depend on the viral system and target cell line used (7, 35, 36, 60). Although the B.1.351 variant has now been displaced by the Delta variant, our results illustrate the need to understand immunity elicited by different SARS-CoV-2 variants. As population immunity due to infection or vaccination increases, preexisting immunity is becoming an increasingly important driver of SARS-CoV-2 evolution (61), as has shown to be the case for seasonal coronaviruses (62, 63). Moreover, as individuals begin to accumulate more complex SARS-CoV-2 immune histories due to multiple infections and/or vaccinations, the effects of immune imprinting or original antigenic sin (64, 65) may start to interact with the variant-specific immunodominance hierarchies we have describ...

    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 10.1101/2021.10.12.464114v1 on bioRxiv