Highly Specific Memory B Cells Generation after the 2nd Dose of BNT162b2 Vaccine Compensate for the Decline of Serum Antibodies and Absence of Mucosal IgA

  1. Eva Piano Mortari
  2. Cristina Russo
  3. Maria Rosaria Vinci
  4. Sara Terreri
  5. Ane Fernandez Salinas
  6. Livia Piccioni
  7. Claudia Alteri
  8. Luna Colagrossi
  9. Luana Coltella
  10. Stefania Ranno
  11. Giulia Linardos
  12. Marilena Agosta
  13. Christian Albano
  14. Chiara Agrati
  15. Concetta Castilletti
  16. Silvia Meschi
  17. Paolo Romania
  18. Giuseppe Roscilli
  19. Emiliano Pavoni
  20. Vincenzo Camisa
  21. Annapaola Santoro
  22. Rita Brugaletta
  23. Nicola Magnavita
  24. Alessandra Ruggiero
  25. Nicola Cotugno
  26. Donato Amodio
  27. Marta Luisa Ciofi Degli Atti
  28. Daniela Giorgio
  29. Nicoletta Russo
  30. Guglielmo Salvatori
  31. Tiziana Corsetti
  32. Franco Locatelli
  33. Carlo Federico Perno
  34. Salvatore Zaffina
  35. Rita Carsetti

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Abstract

Specific memory B cells and antibodies are a reliable read-out of vaccine efficacy. We analysed these biomarkers after one and two doses of BNT162b2 vaccine. The second dose significantly increases the level of highly specific memory B cells and antibodies. Two months after the second dose, specific antibody levels decline, but highly specific memory B cells continue to increase, thus predicting a sustained protection from COVID-19. We show that although mucosal IgA is not induced by the vaccination, memory B cells migrate in response to inflammation and secrete IgA at mucosal sites. We show that the first vaccine dose may lead to an insufficient number of highly specific memory B cells and low concentration of serum antibodies, thus leaving vaccinees without the immune robustness needed to ensure viral elimination and herd immunity. We also clarify that the reduction of serum antibodies does not diminish the force and duration of the immune protection induced by vaccination. The vaccine does not induce sterilizing immunity. Infection after vaccination may be caused by the lack of local preventive immunity because of the absence of mucosal IgA.

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  1. Version published to 10.3390/cells10102541
  2. ScreenIT

    SciScore for 10.1101/2021.06.08.21258284: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethical Approval: Ethical approval was obtained from the Ethics Committee at OPBG, Bambino Gesù Children Hospital.
    Consent: According to the guidelines on Italian observational studies as established by the Italian legislation about the obligatory occupational surveillance and privacy management; Health Care Wokers (HCWs) confidentiality was safeguarded, and informed consent was obtained from all the participants.
    Sex as a biological variablenot detected.
    RandomizationGiven the limiting amount of blood available for the study, we randomly selected 74 HCWs samples for the ELISpot and 34 for FACs (Table 2).
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Wells were coated with 50 µl PBS containing either isotype-specific AffiniPure F(ab’)2 Fragment Goat anti-Human antibody (Jackson ImmunoResearch Laboratories) (anti-IgM final concentration 2.5 µg/mL, anti-IgG 15 µg/mL, anti-IgA 10 µg/mL); or Trimeric Spike at a final concentration of 1 µg/ml to detect specific responses.
    anti-Human
    suggested: None
    anti-IgM
    suggested: None
    anti-IgG
    suggested: None
    Cells were discarded and after washing with PBS + 0,05% Tween (5 x 200 µl/well), plates were incubated for 1 h at 37 °C with peroxidase-conjugated goat anti-human IgM, IgG or IgA antibodies (Jackson ImmunoResearch Laboratories) diluted in PBS + 0,05% Tween + 1% BSA.
    anti-human IgM , IgG
    suggested: None
    IgA
    suggested: None
    ELISA for specific IgA and IgM detection: A semi-quantitative in vitro determination of human IgA antibodies against the SARS-CoV-2 was evaluated on serum, saliva and breast milk samples by using the Anti-SARS-CoV-2 ELISA (Euroimmun), according to the manufacturer’s instructions.
    human IgA
    suggested: None
    Anti-SARS-CoV-2 ELISA ( Euroimmun)
    suggested: None
    After washing again, plates were incubated for 1 h at 37°C with peroxidase-conjugated goat anti-human IgM antibody (Jacksons ImmunoResearch Laboratories).
    anti-human IgM
    suggested: None
    Absorbance at 450 nm was measured, and IgM concentrations were calculated by interpolation from the standard curve based on serial dilutions of monoclonal human IgM antibody against SARS-CoV-2 Spike-RBD (Invivogen)43.
    SARS-CoV-2
    suggested: None
    Spike-RBD
    suggested: None
    Quantitative determination of anti-N, anti-S, Trimeric Spike and RBD antibodies: Serum samples were tested by two different methods and analytical platforms.
    anti-N
    suggested: None
    anti-S
    suggested: None
    Qualitative detection of antibodies direct against the nucleocapsid (N) protein and semi-quantitative detection of total antibodies (IgA, IgM, IgG) directed against the RBD of the virus Spike (S) protein of SARSxxxxx-CoV-2 were tested by an electro-chemiluminescence sandwich immunoassay (ECLIA), using Elecsys-anti SARS-CoV-2 and Elecsys-anti SARS-CoV-2 S (Roche Diagnostics) test on a Cobas e801 analyzer following the manufacturer’s instructions.
    IgA , IgM , IgG
    suggested: None
    Elecsys-anti SARS-CoV-2
    suggested: None
    Elecsys-anti SARS-CoV-2 S
    suggested: None
    The quantitative determination of anti-Trimeric spike protein specific IgG antibodies to SARS-CoV-2 was run on LiaisonXL platform by a new generation of chemiluminescence immunoassay (CLIA) TrimericS IgG assay (DiaSorin) with a quantification range between 4.81 BAU/mL and 2080 BAU/mL (dilution factor 1:20).
    anti-Trimeric spike protein specific IgG
    suggested: None
    The antigen probes prepared individually as above were then mixed in Brilliant Buffer (BD). ∼5x106 previously frozen PBMC samples were prepared and stained with 85μL antigen probe cocktail containing 100ng Spike per probe (total 200ng), 27.5ng RBD and 20ng SA-PE-Cy7 at 4°C for 30min to ensure maximal staining quality before surface staining with antibodies as listed in table S1 was performed in Brilliant Buffer at 4°C for 30min.
    30min
    suggested: None
    Software and Algorithms
    SentencesResources
    The SARS-CoV-2 IgG II kit, a chemiluminescence microparticle antibody assays detecting antibodies against the RBD of SARS-CoV-2, (CMIA, IgG antibody concentrations expressed as arbitrary units, AU/mL ≥ 50 are considered positive, ARCHITECT® i2000s Abbott Diagnostics) was used according to manufacturer’s protocols.
    Abbott
    suggested: (Abbott, RRID:SCR_010477)
    Statistical analyses were performed with GraphPad Prism 8.0 (GraphPad Software).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 40, 38 and 39. 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.
    • Thank you for including a protocol registration statement.

    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.

  3. Version published to 10.1101/2021.06.08.21258284v1 on medRxiv