COVID-19 mRNA third dose induces a unique hybrid immunity-like antibody response

  1. Emanuele Andreano
  2. Ida Paciello
  3. Giulio Pierleoni
  4. Giulia Piccini
  5. Valentina Abbiento
  6. Giada Antonelli
  7. Piero Pileri
  8. Noemi Manganaro
  9. Elisa Pantano
  10. Giuseppe Maccari
  11. Silvia Marchese
  12. Lorena Donnici
  13. Linda Benincasa
  14. Ginevra Giglioli
  15. Margherita Leonardi
  16. Concetta De Santi
  17. Massimiliano Fabbiani
  18. Ilaria Rancan
  19. Mario Tumbarello
  20. Francesca Montagnani
  21. Claudia Sala
  22. Duccio Medini
  23. Raffaele De Francesco
  24. Emanuele Montomoli
  25. Rino Rappuoli

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Abstract

The continuous evolution of SARS-CoV-2 generated highly mutated variants, like omicron BA.1 and BA.2, able to escape natural and vaccine-induced primary immunity 1,2 . The administration of a third dose of mRNA vaccines induces a secondary response with increased protection. We investigated, at single-cell level, the longitudinal evolution of the neutralizing antibody response in four donors after three mRNA doses 3 . A total of 4,100 spike protein specific memory B cells were single cell sorted and 350 neutralizing antibodies were identified. The third dose increased the antibody neutralization potency and breadth against all SARS-CoV-2 variants of concern as previously observed with hybrid immunity 3 . However, the B cell repertoire that stands behind the response is dramatically different. The increased neutralizing response was largely due to the expansion of B cell germlines poorly represented after two doses, and the reduction of germlines predominant after primary immunization such as IGHV3-53;IGHJ6-1 and IGHV3-66;IGHJ4-1. Divergently to hybrid immunity, cross-protection after a third dose was mainly guided by Class 1/2 antibodies encoded by IGHV1-58;IGHJ3-1 and IGHV1-69;IGHJ4-1 germlines. The IGHV2-5;IGHJ3-1 germline, which induced broadly cross-reactive Class 3 antibodies after infection or viral vector vaccination, was not induced by a third mRNA dose. Our data show that while neutralizing breadth and potency can be improved by different immunization regimens, each of them has a unique molecular signature which should be considered while designing novel vaccines and immunization strategies.

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  1. ScreenIT

    SciScore for 10.1101/2022.05.09.491201: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Enrollment of COVID-19 vaccinees and human sample collection: This work results from a collaboration with the Azienda Ospedaliera Universitaria Senese, Siena (IT) that provided samples from COVID-19 vaccinated donors, of both sexes, who gave their written consent.
    IRB: The study was approved by the Comitato Etico di Area Vasta Sud Est (CEAVSE) ethics committees
    Sex as a biological variableEnrollment of COVID-19 vaccinees and human sample collection: This work results from a collaboration with the Azienda Ospedaliera Universitaria Senese, Siena (IT) that provided samples from COVID-19 vaccinated donors, of both sexes, who gave their written consent.
    RandomizationThis study was unblinded and not randomized.
    Blindingnot detected.
    Power AnalysisNo statistical methods were used to predetermine sample size.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-Human IgG –Peroxidase antibody (Fab specific) produced in goat (Sigma) diluted 1:45000 in sample buffer was then added and samples incubated for 1 h at 37°C.
    Anti-Human IgG –Peroxidase antibody
    suggested: None
    Anti-Human IgG
    suggested: (Abcam Cat# ab45000, RRID:AB_732293)
    Experimental Models: Cell Lines
    SentencesResources
    The mixture was then added to the wells of a 96-well plate containing a sub-confluent Vero E6 cell monolayer.
    Vero E6
    suggested: RRID:CVCL_XD71)
    HEK293TN-hACE2 cell line generation: HEK293TN-hACE2 cell line was generated by lentiviral transduction of HEK293TN (System Bioscience) cells as described in Notarbartolo S. et al25
    HEK293TN-hACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    The transfer vector pLENTI_hACE2_HygR was obtained by cloning of hACE2 from pcDNA3.1-hACE2 (Addgene #145033) into pLenti-CMV-GFP-Hygro (Addgene #17446).
    pLENTI_hACE2_HygR
    suggested: RRID:Addgene_155296)
    pcDNA3.1-hACE2
    suggested: RRID:Addgene_145033)
    pLenti-CMV-GFP-Hygro
    suggested: None
    Production of SARS-CoV-1 pseudoparticles: SARS-CoV-1 lentiviral pseudotype particles were generated as described in Conforti et al. for SARS-CoV-226 by using the SARS-CoV1 SPIKE plasmid pcDNA3.3_CoV1_D28 (
    SPIKE
    suggested: None
    pcDNA3.3_CoV1_D28
    suggested: RRID:Addgene_170447)
    Software and Algorithms
    SentencesResources
    FACSDiva
    FACSDiva
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    Software (version 9) was used for data acquisition and analysis was performed using FlowJo (version 10).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Obtained relative light units (RLUs) were normalized to controls and dose response curve were generated by nonlinear regression curve fitting with GraphPad Prism to calculate Neutralization Dose 50 (ND50).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    A network graph was generated with the R package ggraph v2.0.5 (https://ggraph.data-imaginist.com/index.html) with Fruchterman-Reingold layout algorithm and the figure was assembled with ggplot2 v3.3.5.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    GraphPad Software, Inc.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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.

    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 10.1101/2022.05.09.491201v1 on bioRxiv