Screening HLA-A-restricted T cell epitopes of SARS-CoV-2 and the induction of CD8+ T cell responses in HLA-A transgenic mice

  1. Xiaoxiao Jin
  2. Yan Ding
  3. Shihui Sun
  4. Xinyi Wang
  5. Zining Zhou
  6. Xiaotao Liu
  7. Miaomiao Li
  8. Xian Chen
  9. Anran Shen
  10. Yandan Wu
  11. Bicheng Liu
  12. Jianqiong Zhang
  13. Jian Li
  14. Yi Yang
  15. Haibo Qiu
  16. Chuanlai Shen
  17. Yuxian He
  18. Guangyu Zhao

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Abstract

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific T cells have been found to play essential roles in host immune protection and pathology in patients with coronavirus disease 2019 (COVID-19), this study focused on the functional validation of T cell epitopes and the development of vaccines that induce specific T cell responses. A total of 120 CD8 + T cell epitopes from the E, M, N, S, and RdRp proteins were functionally validated. Among these, 110, 15, 6, 14, and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; in addition, four epitopes from the S protein displayed one amino acid that was distinct from the current SARS-CoV-2 variants. Then, 31 epitopes restricted by the HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or poly (lactic-co-glycolic acid) nanoparticles, and these vaccines elicited robust and specific CD8 + T cell responses in HLA-A2/DR1 transgenic mice as well as wild-type mice. In contrast to previous research, this study established a modified DC-peptide-PBL cell coculture system using healthy donor PBMCs to validate the in silico predicted epitopes, provided an epitope library restricted by nine of the most prevalent HLA-A allotypes covering broad Asian populations, and identified the HLA-A restrictions of these validated epitopes using competitive peptide binding experiments with HMy2.CIR cell lines expressing the indicated HLA-A allotype, which initially confirmed the in vivo feasibility of 9- or 10-mer peptide cocktail vaccines against SARS-CoV-2. These data will facilitate the design and development of vaccines that induce antiviral CD8 + T cell responses in COVID-19 patients.

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  1. Version published to 10.1038/s41423-021-00784-8
  2. Version published to 10.1101/2021.04.01.438020v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.04.01.438020: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The human samples collection and use has been approved by Clinical Ethics Committee of Affiliated Zhongda hospital of Southeast University. 2. Mice: Female HLA-A*02:01/DR1 transgenic and H-2-β2m-/-/IAβ-/- C57BL/6 mice at 10 weeks were generous gifts from Academy of Military Medical Sciences.
    IACUC: Animal welfare and experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and were approved by the Animal Ethics Committee of Southeast University. 3.
    RandomizationTwelve female HLA-A2/DR1 transgenic mice were randomly divided into four groups.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableFemale C57BL/6 mice at 10 weeks of age were purchased from the Comparative Medicine Center of Yangzhou University (Yangzhou, China).

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were then harvested, washed, blocked with anti-mouse CD16/CD32 for 20 min at 4°C and were stained with FITC-labeled anti-CD3 and APC-labeled anti-CD8 antibodies for 30 min at 4 °C.
    anti-mouse CD16/CD32
    suggested: None
    At day 22, cells were harvested and blocked with anti-mouse CD16/CD32 for 20 min, then stained with PE-labeled anti-CD3 and APC-labeled anti-CD8 antibodies for 30 min at 4°C for further analysis on the flow cytometry.
    anti-CD3
    suggested: None
    Then the cell lines were stained with fluorescence-labeled monoclonal antibody W6/32 against HLA-ABC or anti-HLA-A24, the high-expression cells were then sorted using flow cytometry and followed by pure culture and sequencing analyses.
    HLA-ABC
    suggested: None
    anti-HLA-A24
    suggested: None
    Then T2 cells were stained with PE-labeled anti-HLA-A2.1 antibody for 30 min at 4 °C followed by flow cytometry.
    anti-HLA-A2.1
    suggested: None
    After incubation for 20 h at 37 ℃ and 5% CO2, the plates were washed and then incubated with biotinylated anti-IFN-γ detecting antibody (BD) for 2h at RT.
    anti-IFN-γ
    suggested: None
    Cells were then harvested, washed, blocked with anti-mouse CD16/CD32 for 20 minutes at 4°C and were stained with FITC-labeled anti-CD3 and APC-labeled anti-CD8 antibodies for 30 min at 4 °C.
    anti-CD8
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    The human samples collection and use has been approved by Clinical Ethics Committee of Affiliated Zhongda hospital of Southeast University. 2. Mice: Female HLA-A*02:01/DR1 transgenic and H-2-β2m-/-/IAβ-/- C57BL/6 mice at 10 weeks were generous gifts from Academy of Military Medical Sciences.
    HLA-A*02:01/DR1
    suggested: None
    Female C57BL/6 mice at 10 weeks of age were purchased from the Comparative Medicine Center of Yangzhou University (Yangzhou, China).
    C57BL/6
    suggested: None
    Twelve female HLA-A2/DR1 transgenic mice were randomly divided into four groups.
    HLA-A2/DR1
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04546841RecruitingSafety and Immunogenicity Trial of Multi-peptide Vaccination…

    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 49, 43 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.

  4. Version published to 10.1101/2021.04.01.438020v1 on bioRxiv