Immunogenic epitope panel for accurate detection of non-cross-reactive T cell response to SARS-CoV-2

  1. Aleksei Titov
  2. Regina Shaykhutdinova
  3. Olga V. Shcherbakova
  4. Yana V. Serdyuk
  5. Savely A. Sheetikov
  6. Ksenia V. Zornikova
  7. Alexandra V. Maleeva
  8. Alexandra Khmelevskaya
  9. Dmitry V. Dianov
  10. Naina T. Shakirova
  11. Dmitry B. Malko
  12. Maxim Shkurnikov
  13. Stepan Nersisyan
  14. Alexander Tonevitsky
  15. Ekaterina Khamaganova
  16. Anton V. Ershov
  17. Elena Y. Osipova
  18. Ruslan V. Nikolaev
  19. Dmitry E. Pershin
  20. Viktoria A. Vedmedskia
  21. Michael Maschan
  22. Victoria R. Ginanova
  23. Grigory A. Efimov

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Abstract

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  1. Version published to 10.1172/jci.insight.157699
  2. ScreenIT

    SciScore for 10.1101/2021.12.12.21267518: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: The clinical trial of the Corona-T-test kit was conducted at Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology in Moscow, Russia.
    Consent: Written informed consent was obtained from all patients.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    BlindingDonors were recruited by a separate center, DNKOM Laboratory, where the samples were collected and blinded.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Vac PBMCs (n = 45) were collected and frozen 23–65 days after receiving the second dose of the Sputnik-V (Gam-COVID-Vac) vaccine from donors who had a negative antibody tests no later than four weeks before the first shot and no self-reported COVID-like symptoms.
    Gam-COVID-Vac
    suggested: None
    The HE cohort (n = 37) of samples were from people who had close contact with a patient with active COVID-19 (same household or “red zone” medical workers with multiple negative RT-PCR tests) but who were themselves without COVID-19 symptoms and without detectable IgG and IgM anti-S protein antibodies.
    IgM anti-S protein
    suggested: (Creative Diagnostics Cat# DMABT-Z60199, RRID:AB_2455687)
    All samples were pretested for antibodies against SARS-CoV-2, and inclusion criteria and antibody tests are shown in the table below.
    SARS-CoV-2
    suggested: None
    The plates were washed thrice with PBST, and then 100 µL of 0.1 µg/mL biotinylated anti-IFNɣ antibody (R&D Systems, USA) was added and incubated for one hour at 37°C on a rocking platform.
    anti-IFNɣ
    suggested: None
    Anti-SARS-CoV-2 ELISA: ELISA kits for the detection of anti-RBD IgG (K153, National Research Center for Hematology, Russia) and SARS-CoV-2-IgМ-EIA-BEST (D-5502, Vector Best, Russia) for the detection of IgM antibodies to full-length S protein were used according to the manufacturers’ instructions.
    anti-RBD IgG
    suggested: None
    K153
    suggested: (Leinco Technologies Cat# K153, RRID:AB_2893857)
    SARS-CoV-2-IgМ-EIA-BEST
    suggested: None
    D-5502 , Vector Best ,
    suggested: None
    IgM
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    HLA and epitope selection: We selected the HLA list based on the most-presented HLA among the CP cohort: HLA-I: A*01:01; A*02:01; A*03:01; A*11:01; A*23:01; A*24:02; A*25:01; A*30:01; A*32:01; B*07:02; B*08:01; B*13:02; B*15:01; B*18:01; B*27:05; B*35:01; B*38:01; B*40:01; B*44:02; B*44:03; B*51:01; C*01:02; C*03:04; C*04:01; C*05:01; C*06:02; C*07:01; C*07:02; C*12:03; C*15:02 MHC-I peptides: The core set comprised two epitopes from Shomuradova et al. and 25 minimally/non-cross-reactive epitopes from the publications listed in Fig 1A. 67 epitopes from Snyder et al. were chosen with the aim of selecting individual epitopes instead of peptides within a peptide pool and balancing predicted HLA coverage, minimal cross-reactivity (based on the number of non-naive expansions from healthy donors assigned to a specific epitope/peptide group), and higher immunogenicity (based on the number of CP with detected TCR sequences assigned to a specific epitope/peptide group).
    B*08:01; B*13:02; B*15:01; B*18:01; B*27:05; B*35:01; B*38:01; B*40:01; B*44:02; B*44:03
    suggested: None
    Software and Algorithms
    SentencesResources
    Meikang Biological Project, KH-M-02, China) were coated with 100 μL per well of 0.01 mg/mL anti-IFNɣ antibody (Hytest, clone GF1) in 100 mM bicarbonate/carbonate (pH 9.6).
    Meikang Biological Project
    suggested: None
    Alignments were performed using best global-local alignment by the ‘pairwiseAlignment’ (bioPython) function for four ORFs shared by all five strains: orf1ab, S, N, and M, allowing amino acid substitutions with similar biochemical properties (1, 2) and low penalties for gap opening and extension (−0.5).
    bioPython
    suggested: (Biopython, RRID:SCR_007173)
    We used the following mixes in our work (each presented by two submixes, diluted in DMSO or isopropanol): Quantification and statistical analysis: All data comparisons were performed using GraphPad Prism 8, Python 3.2, and FlowJo 10 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Python
    suggested: (IPython, RRID:SCR_001658)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Fixation and permeabilization were performed with BD Cytofix/Cytoperm according to the manufacturer’s protocol, and intracellular staining was carried out for 30 min in the dark at room temperature with APC anti-IFNɣ (clone B27, BD Biosciences, USA).
    BD Cytofix/Cytoperm
    suggested: None

    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.

  3. Version published to 10.1101/2021.12.12.21267518v1 on medRxiv