BNT162b2 induces SARS-CoV-2-neutralising antibodies and T cells in humans

  1. Ugur Sahin
  2. Alexander Muik
  3. Isabel Vogler
  4. Evelyna Derhovanessian
  5. Lena M. Kranz
  6. Mathias Vormehr
  7. Jasmin Quandt
  8. Nicole Bidmon
  9. Alexander Ulges
  10. Alina Baum
  11. Kristen Pascal
  12. Daniel Maurus
  13. Sebastian Brachtendorf
  14. Verena Lörks
  15. Julian Sikorski
  16. Peter Koch
  17. Rolf Hilker
  18. Dirk Becker
  19. Ann-Kathrin Eller
  20. Jan Grützner
  21. Manuel Tonigold
  22. Carsten Boesler
  23. Corinna Rosenbaum
  24. Ludwig Heesen
  25. Marie-Cristine Kühnle
  26. Asaf Poran
  27. Jesse Z. Dong
  28. Ulrich Luxemburger
  29. Alexandra Kemmer-Brück
  30. David Langer
  31. Martin Bexon
  32. Stefanie Bolte
  33. Tania Palanche
  34. Armin Schultz
  35. Sybille Baumann
  36. Azita J. Mahiny
  37. Gábor Boros
  38. Jonas Reinholz
  39. Gábor T. Szabó
  40. Katalin Karikó
  41. Pei-Yong Shi
  42. Camila Fontes-Garfias
  43. John L. Perez
  44. Mark Cutler
  45. David Cooper
  46. Christos A. Kyratsous
  47. Philip R. Dormitzer
  48. Kathrin U. Jansen
  49. Özlem Türeci

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Abstract

BNT162b2, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA (mRNA) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) stabilized in the prefusion conformation, has demonstrated 95% efficacy to prevent coronavirus disease 2019 (COVID-19). Recently, we reported preliminary BNT162b2 safety and antibody response data from an ongoing placebo-controlled, observer-blinded phase 1/2 vaccine trial 1 . We present here antibody and T cell responses from a second, non-randomized open-label phase 1/2 trial in healthy adults, 19-55 years of age, after BNT162b2 prime/boost vaccination at 1 to 30 µg dose levels. BNT162b2 elicited strong antibody responses, with S-binding IgG concentrations above those in a COVID-19 human convalescent sample (HCS) panel. Day 29 (7 days post-boost) SARS-CoV-2 serum 50% neutralising geometric mean titers were 0.3-fold (1 µg) to 3.3-fold (30 µg) those of the HCS panel. The BNT162b2-elicited sera neutralised pseudoviruses with diverse SARS-CoV-2 S variants. Concurrently, in most participants, S-specific CD8 + and T helper type 1 (T H 1) CD4 + T cells had expanded, with a high fraction producing interferon-γ (IFNγ). Using peptide MHC multimers, the epitopes recognised by several BNT162b2-induced CD8 + T cells when presented on frequent MHC alleles were identified. CD8 + T cells were shown to be of the early-differentiated effector-memory phenotype, with single specificities reaching 0.01-3% of circulating CD8 + T cells. In summary, vaccination with BNT162b2 at well tolerated doses elicits a combined adaptive humoral and cellular immune response, which together may contribute to protection against COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.09.20245175: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The trial was carried out in Germany in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines and with approval by an independent ethics committee (Ethik-Kommission of the Landesärztekammer Baden-Württemberg, Stuttgart, Germany) and the competent regulatory authority (Paul-Ehrlich Institute, Langen, Germany).
    Consent: All participants provided written informed consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableClinical trial design: Study BNT162-01 (NCT04380701) is an ongoing, umbrella-type first-in-human, phase 1/2, open-label, dose-ranging clinical trial to assesses the safety, tolerability, and immunogenicity of ascending dose levels of various intramuscularly administered BNT162 mRNA vaccine candidates in healthy men and non-pregnant women 18 to 55 years (amended to add 56-85 years) of age.
    Cell Line AuthenticationContamination: Cell lines were tested for mycoplasma contamination after receipt and before expansion and cryopreservation.

    Table 2: Resources

    Antibodies
    SentencesResources
    A secondary fluorescently labelled goat anti-human polyclonal antibody (Jackson Labs) was added for 90 minutes at room temperature while shaking, before plates were washed once more in a solution containing 0.05% Tween-20.
    A secondary fluorescently labelled goat anti-human polyclonal antibody ( Jackson Labs )
    suggested: None
    anti-human polyclonal antibody
    suggested: (LSBio (LifeSpan Cat# LS-C6444-30, RRID:AB_860431)
    To account for varying sample quality reflected in the number of spots in response to anti-CD3 antibody stimulation, a normalisation method was applied, enabling direct comparison of spot counts and strength of response between individuals.
    anti-CD3
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Viral master stocks (2 × 107 PFU/mL) were grown in Vero E6 cells as previously described38.
    Vero E6
    suggested: None
    Serial dilutions of heat-inactivated sera were incubated with the reporter virus (2 × 104 PFU per well to yield a 10-30% infection rate of the Vero CCL81 monolayer) for 1 hour at 37 °C before inoculating Vero CCL81 cell monolayers (targeted to have 8,000 to 15,000 cells in a central field of each well at the time of seeding, 24 hours before infection) in 96-well plates to allow accurate quantification of infected cells.
    Vero CCL81
    suggested: None
    HEK293T cells (ATCC CRL-3216) were seeded (culture medium: DMEM high glucose [Life Technologies] supplemented with 10% heat-inactivated FBS (Life Technologies) and penicillin/streptomycin/L-glutamine [Life Technologies]) and transfected the following day with S expression plasmid using Lipofectamine LTX (Life Technologies) following the manufacturer’s protocol.
    HEK293T
    suggested: None
    Serum dilutions were mixed 1:1 with pseudoparticles for 30 minutes at room temperature prior to addition to Vero cells and incubation at 37 °C for 24 hours.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    Key exclusion criteria included previous clinical or microbiological diagnosis of COVID-19; receipt of medications to prevent COVID-19; previous vaccination with any coronavirus vaccine; a positive serological test for SARS-CoV-2 IgM and/or IgG; and a SARS-CoV-2 nucleic acid amplification test (NAAT)-positive nasal swab; increased risk for severe COVID-19; and immunocompromised individuals.
    IgG
    suggested: (DSHB Cat# LEP100 IgG, RRID:AB_528124)
    Total cell counts per well were enumerated by nuclear stain (Hoechst 33342) and fluorescent virally infected foci were detected 16-24 hours after inoculation with a Cytation 7 Cell Imaging Multi-Mode Reader (BioTek) with Gen5 Image Prime version 3.09.
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    FlowJo LLC, BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Surface and viability staining was carried out in flow buffer (DPBS [Gibco] with 2% FBS [Biochrom], 2 mM EDTA [Sigma-Aldrich]) supplemented with BSB Plus for 30 minutes at 4 °C (CD3 BUV395, 1:50; CD45RA BUV563, 1:200; CD27 BUV737, 1:200; CD8 BV480, 1:200; CD279 BV650, 1:20; CD197 BV786, 1:15; CD4 BB515, 1:50; CD28 BB700, 1:100; CD38 PE-CF594, 1:600; HLA-DR APC-R700, 1:150; all BD Biosciences; DUMP channel: CD14 APC-eFluor780, 1:100; CD16 APC-eFluor780, 1:100; CD19 APC-eFluor780, 1:100; fixable viability dye eFluor780, 1:1,667; all ThermoFisher Scientific).
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    All statistical analyses were performed using GraphPad Prism software version 8.4.2.
    GraphPad Prism
    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: We detected the following sentences addressing limitations in the study:
    Limitations of our clinical study include the small sample size, the lack of representation of populations of interest (e.g. older adults, other ethnicities, immunocompromised individuals and pediatric populations), and limited availability of blood samples for a more in-depth T cell analysis. Typically antigen-activated B and T cells go through proliferation, followed by rebound contraction with a gradual decline in numbers before entering a sustained memory phase22,23, and the short-term follow up presented in this paper does not allow for extrapolation of long-term durability of the immune responses. Whereas it is encouraging that BNT162b2 robustly activates antigen-specific humoral and as cellular immune effector systems, it is not clear whether this immune response pattern will protect from SARS-CoV-2 infection and prevent COVID-19. These questions will be addressed by the ongoing clinical program, which includes longer term follow-up of participants in the two ongoing phase 1/2 trials, a dedicated immune biomarker trial to further dissect the composite elements of the immune response, and the ongoing phase 2/3 study with efficacy endpoints.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04380701RecruitingA Trial Investigating the Safety and Effects of Four BNT162 …
    NCT04368728Active, not recruitingStudy to Describe the Safety, Tolerability, Immunogenicity, …

    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 36 and 44. 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.
    • No protocol registration statement was detected.

    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. NCRC

    Our take

    This study was available as a preprint and thus was not yet peer reviewed. In-depth characterization of vaccine-induced adaptive immune response is a critical step in developing our understanding of COVID-19 immunity. A very thorough evaluation of both the humoral and cellular immune responses elicited by the Pfizer/BioNTech BNT162b2 mRNA COVID-19 vaccine is presented here and provides largely encouraging data. However, the authors’ cursory assessment of the vaccine’s efficacy against the SARS-CoV-2 variants provides little reassurance, and should be largely disregarded. Although no evidence suggests that this vaccine’s efficacy would be reduced against any SARS-CoV-2 variant, the data presented here are insufficient to speak meaningfully to this topic.

    Study design

    non-randomized-trial

    Study population and setting

    This report presents data from a non-randomized open-label phase 1/2 trial evaluating humoral and cellular immune responses elicited in humans from the Pfizer/BioNTech BNT162b2 mRNA COVID-19 vaccine, which encodes the entirety of SARS-CoV-2 spike (S) protein. The total study population consisted of 48 white individuals (21 male, 27 female) aged 19-55 years, and each participant received two doses of the vaccine 21 days apart in a prime-boost regimen, with each dose containing 1, 10, 20, or 30 ug mRNA (NB: the approved Pfizer/BioNTech vaccine product currently in use utilizes a 30 ug dose of mRNA for each dose). Total SARS-CoV-2 specific antibody levels, neutralizing antibody titers, and cellular immune responses were measured at various timepoints. Additionally, sera from vaccinated participants were evaluated for neutralization of pseudotype vesicular stomatitis virus (VSV) expressing S from different SARS-CoV-2 variants. Participants were monitored for adverse reactions.

    Summary of main findings

    Increases in total SARS-CoV-2 specific antibody levels were observed following both the first and second doses of the vaccine, with particularly robust responses noted following the boost. All doses above 10 ug elicited a similar response, and levels two months post-boost amongst these doses exceeded those observed in convalescent COVID-19 patients. Neutralizing antibody titers increased substantially only following the boost, with a strong dose-dependent response observed for the 10, 20, and 30 ug doses (the 1 ug dose did not elicit an appreciable neutralizing antibody response). Two months following boost, neutralizing antibody titers amongst vaccinated participants also remained elevated above titers observed in convalescent COVID-19 patients. Neutralizing titers against the SARS-CoV-2 variants assessed using the pseudotype VSV assay were similar to those observed against the reference virus used, which was obtained from a COVID-19 patient in Washington state in January 2020 (SARS-CoV-2 USA_WA1/2020). Measurable CD4+ and CD8+ T-cell responses were elicited against multiple epitopes throughout S (i.e., not just within the receptor binding domain) in 32/34 and 34/37 participants analyzed, respectively, and were not clearly dose-dependent above 10 ug. Specific epitopes recognized by vaccine-induced T-cells were also identified. A TH1-predominant response was reported and reduces concern regarding the possibility of vaccine-induced disease enhancement. Finally, the strength of the cellular immune response was noted to correlate with the strength of humoral immune response within individuals. Consistent with previous reports, no serious adverse reactions were recorded.

    Study strengths

    This study provides an in-depth analysis (much of which is beyond the scope of this summary) of the adaptive immune response elicited by a COVID-19 vaccine currently in wide use. Particular attention is given to characterizing T-cell responses, providing data that will likely prove valuable for numerous ongoing and future studies. Additionally, SARS-CoV-2 specific antibody levels and neutralizing antibody titers are evaluated out to approximately 2 months (63 days) post-boost, extending our current scope of knowledge.

    Limitations

    A narrow demographic was utilized for this study, and did not include any elderly or non-white participants. The variant neutralization assay appears to be an afterthought and that aspect of the study is poorly designed. Evidently, the variants utilized each differed from the reference receptor binding domain sequence within S only by a single amino acid, and thus are not at all representative of the variants that are currently of concern in circulation (e.g., B.1.1.7, B.1.351, P.1), which generally harbor mutations at numerous sites within S. Additionally, it seems that there was approximately a log-fold increase in neutralization titers when using the pseudotype VSV assay. Furthermore, as the correlate(s) of protection remain unknown at this time, measurement of only neutralizing antibodies at a single time point for the variant analysis provides extremely limited information regarding overall vaccine efficacy in this context.

    Value added

    This study provides one of the most in-depth characterizations of COVID-19 vaccine-induced adaptive immune responses to date.

  3. Version published to 10.1101/2020.12.09.20245175v1 on medRxiv