An immunodominance hierarchy exists in CD8 + T cell responses to HLA-A*02:01-restricted epitopes identified from the non-structural polyprotein 1a of SARS-CoV-2

  1. Akira Takagi
  2. Masanori Matsui

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Abstract

COVID-19 vaccines are being rapidly developed and human trials are underway. Almost all of these vaccines have been designed to induce antibodies targeting spike protein of SARS-CoV-2 in expectation of neutralizing activities. However, non-neutralizing antibodies are at risk of causing antibody-dependent enhancement. Further, the longevity of SARS-CoV-2-specific antibodies is very short. Therefore, in addition to antibody-induced vaccines, novel vaccines on the basis of SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs) should be considered in the vaccine development. Here, we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Eighty-two peptides were firstly predicted as epitope candidates on bioinformatics. Fifty-four in 82 peptides showed high or medium binding affinities to HLA-A*02:01. HLA-A*02:01 transgenic mice were then immunized with each of the 54 peptides encapsulated into liposomes. The intracellular cytokine staining assay revealed that 18 out of 54 peptides were CTL epitopes because of the induction of IFN-γ-producing CD8 + T cells. In the 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant CTL epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant over the other peptides. Surprisingly, all mice immunized with the liposomal 10 peptide mixture did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.

Importance

For the development of vaccines based on SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs), we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Out of 82 peptides predicted on bioinformatics, 54 peptides showed good binding affinities to HLA-A*02:01. Using HLA-A*02:01 transgenic mice, 18 in 54 peptides were found to be CTL epitopes in the intracellular cytokine staining assay. Out of 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant. Surprisingly, all immunized mice did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.18.304493: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: This study was approved by the Animal Research Committee of Saitama Medical University.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After 3 hours’ incubation at 37°C, peptide-pulsed cells were stained with anti-HLA-A2 monoclonal antibody, BB7.2 (47), followed by FITC-labeled goat anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO).
    anti-HLA-A2
    suggested: None
    anti-mouse IgG
    suggested: None
    In brief, after 1 wk following immunization, spleen cells were incubated with 50 μM of each peptide for 5 hours at 37°C in the presence of brefeldin A (GolgiPlug™, BD Biosciences), and were stained with FITC-conjugated anti-mouse CD8 monoclonal antibody (mAb) (BioLegend, San Diego, CA).
    anti-mouse CD8
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, RMA-S-HHD cells were pre-cultured overnight at 26°C in a CO2 incubator, and then pulsed with each peptide at various concentrations ranging from 0.01 μM to 100 μM for 1 hour at 26°C.
    RMA-S-HHD
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: We used HLA-A*02:01 transgenic mice (37) that express a transgenic HLA-A*02:01 monochain, designated as HHD, in which human β2-microglobulin is covalently linked to a chimeric heavy chain composed of HLA-A*02:01 (α1 and α2 domains) and H-2Db (α3, transmembrane, and cytoplasmic domains).
    HLA-A*02:01
    suggested: RRID:IMSR_TAC:9659)
    Software and Algorithms
    SentencesResources
    Prediction of CTL epitopes: Four computer-based programs including SYFPEITHI (31), nHLAPred (32), ProPred-I (33), and IEDB (34) were used to predict HLA-A*02:01-restricted CTL epitopes derived from pp1a of SARS-CoV-2 (GenBank accession numbers: LC528232.1 & LC528233.1).
    SYFPEITHI
    suggested: None
    In brief, after 1 wk following immunization, spleen cells were incubated with 50 μM of each peptide for 5 hours at 37°C in the presence of brefeldin A (GolgiPlug™, BD Biosciences), and were stained with FITC-conjugated anti-mouse CD8 monoclonal antibody (mAb) (BioLegend, San Diego, CA).
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    Statistical analyses: One-way ANOVA followed by post-hoc tests was performed for statistical analyses among multiple groups using Graphpad Prism 5 software (GraphPad software, San Diego, CA).
    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: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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

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  2. Version published to 10.1101/2020.09.18.304493 on bioRxiv