DECREASED BREADTH OF THE ANTIBODY RESPONSE TO THE SPIKE PROTEIN OF SARS-CoV-2 AFTER REPEATED VACCINATION

  1. Lydia Horndler
  2. Pilar Delgado
  3. Salvador Romero-Pinedo
  4. Marina Quesada
  5. Ivaylo Balabanov
  6. Rocío Laguna-Goya
  7. Patricia Almendro-Vázquez
  8. Miguel A. Llamas
  9. Manuel Fresno
  10. Estela Paz-Artal
  11. Hisse M. van Santen
  12. Stela Álvarez
  13. Asunción Olmo
  14. Balbino Alarcón

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Abstract

The rapid development of vaccines to prevent infection by SARS-CoV-2 virus causing COVID-19 makes necessary to compare the capacity of the different vaccines in terms of development of a protective humoral response. Here, we have used a highly sensitive and reliable flow cytometry method to measure the titers of antibodies of the IgG1 isotype in blood of healthy volunteers after receiving one or two doses of the vaccines being administered in Spain. We took advantage of the multiplexed capacity of the method to measure simultaneously the reactivity of antibodies with the S protein of the original strain Wuhan and the variants B.1.1.7 (Alpha), B.1.617.2 (Delta) and B.1.617.1 (Kappa). We found significant differences in the titer of anti-S antibodies produced after a first dose of the vaccines ChAdOx1 nCov-19/AstraZeneca, mRNA-1273/Moderna, BNT162b2/Pfizer-BioNTech and Ad26.COV.S/Janssen. Most important, we found a relative reduction in the reactivity of the sera with the Alpha, Delta and Kappa variants, versus the Wuhan one, after the second boosting immunization. These data allow to make a comparison of different vaccines in terms of anti-S antibody generation and cast doubts about the convenience of repeatedly immunizing with the same S protein sequence.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.12.21261952: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: (HUP) approved by the Research Ethics Committee (no. #4070).
    Consent: All participants provided written consent to participate in the study which was performed according to the EU guidelines and following the ethical principles of the Declaration of Helsinki.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    BlindingSerum samples were received coded from the providers and the experimentalists were blinded to their nature until all data analysis was finalized.
    Power AnalysisSample analysis was carried out in duplicate or triplicate and all experiments were repeated a minimum of two times.
    Cell Line AuthenticationContamination: All cell lines were routinely tested for the absence of mycoplasma.

    Table 2: Resources

    Antibodies
    SentencesResources
    The cell pellet was finally resuspended in a 1:200 dilution of mouse anti-human IgG1 Fc-PE (Ref.: 9054-09, Southern Biotech) and a 1:200 dilution of the Brilliant Violet 421™ anti-human EGFR Antibody (Ref.: 352911, Biolegend) in PBS-BSA.
    anti-human IgG1
    suggested: (SouthernBiotech Cat# 9054-09, RRID:AB_2796628)
    anti-human EGFR
    suggested: (BioLegend Cat# 352911, RRID:AB_2562213)
    Experimental Models: Cell Lines
    SentencesResources
    Human embryonic kidney HEK293T cells (ATCC CRL-3216) were maintained in DMEM supplemented with 10% FBS in a 5% CO2 incubator.
    HEK293T
    suggested: None
    To express the full-length spike S protein of the B.1.1.7 SARS-CoV-2 variant in Jurkat cells (Jurkat-SB117), we used the lentiviral vector based on the epHIV-7 plasmid, the spike S proteins sequences were obtained from the Public Health England database (https://www.gov.uk/coronavirus) and synthesized by the company Eurofins Genomics.
    Jurkat
    suggested: None
    For transduction, lentiviral-transducing supernatants were produced from transfected packaging HEK-293T cells as described previously18.
    HEK-293T
    suggested: None
    Flow cytometry: A 1:1 mix of Jurkat-Swuhan and Jurkat-SB117 cells were incubated for 30 min on ice with 1:50 dilutions of human sera in phosphate-buffered saline (PBS), 1% bovine serum albumin (BSA), 0.02% sodium azide.
    Jurkat-SB117
    suggested: None
    Recombinant DNA
    SentencesResources
    To express the full-length spike S protein of the B.1.1.7 SARS-CoV-2 variant in Jurkat cells (Jurkat-SB117), we used the lentiviral vector based on the epHIV-7 plasmid, the spike S proteins sequences were obtained from the Public Health England database (https://www.gov.uk/coronavirus) and synthesized by the company Eurofins Genomics.
    epHIV-7
    suggested: None
    The EGFP sequence was obtained from the vector pEGFP-C1 (
    pEGFP-C1
    suggested: None
    Briefly, lentiviruses were obtained by co-transfecting plasmids pCMV-dR (gag/pol) and using the JetPEI transfection reagent (Polyplus Transfection).
    pCMV-dR
    suggested: None
    Software and Algorithms
    SentencesResources
    Samples were then washed, labeled with the viability dye 7AAD and analyzed on a FACSCanto II flow cytometer (Becton-Dickinson) and the data were analyzed with FlowJo software (BD).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    All data was analyzed using the GraphPad Prism 7 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Another limitation of our study is that we have compared the reactivity of antibodies with the Wuhan-1 strain and the B.1.1.7 (Alpha) variant and not with the Delta variant which is the most rapidly expanding VOC in Europe and the USA by the end of July 2021 (https://www.cdc.gov/coronavirus/2019-ncov/science/science-briefs/fully-vaccinated-people.html). At present, we are generating a Jurkat-S cell line expressing the Delta variant to confirm that our findings on loss of antibody breadth upon vaccination for the Alpha variant also hold true for the Delta VOC. The generation of this cell line will allow to measure the reactivity of antibodies against the trimeric, native, form of the S protein, something that the serological tests based on the use of recombinant proteins do not allow. While we are awaiting for the results with Jurkat-S expressing the Delta VOC, our results with the Alpha variant suggest that third doses of the present vaccines to the general population might not be the best approach to increase the immunity to the emerging VOCs. In our opinion, a third dose should be limited to the population that has been demonstrated to have been poorly responsive to the first two prime and boost doses.

    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 10.1101/2021.08.12.21261952 on medRxiv