Identification of HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2 and analysis of their conservation using the mutation database of SARS-CoV-2 variants

  1. Akira Takagi
  2. Masanori Matsui

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Abstract

COVID-19 vaccines are currently being administrated worldwide and playing a critical role in controlling the pandemic. They have been designed to elicit neutralizing antibodies against Spike protein of the original SARS-CoV-2, and hence they are less effective against SARS-CoV-2 variants with mutated Spike than the original virus. It is possible that novel variants with abilities of enhanced transmissibility and/or immunoevasion will appear in the near future and perfectly escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8 + cytotoxic T lymphocytes (CTLs). Several lines of evidence suggest the contribution of CTLs on the viral control in COVID-19, and CTLs target a wide range of proteins involving comparatively conserved non-structural proteins. Here, we identified twenty-two HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptide-encapsulated liposomes. We focused on pp1a and HLA-A*24:02 because pp1a is relatively conserved and HLA-A*24:02 is predominant in East Asians such as Japanese. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants. The information of such conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes.

Importance

COVID-19 vaccines have been designed to elicit neutralizing antibodies against the Spike protein of the original SARS-CoV-2, and hence they are less effective against variants. It is possible that novel variants will appear and escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8 + cytotoxic T lymphocytes (CTLs). Here, we identified twenty-two HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a (pp1a) of SARS-CoV-2. We focused on pp1a and HLA-A*24:02 because pp1a is conserved and HLA-A*24:02 is predominant in East Asians. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by mutations in the database of SARS-CoV-2 variants. The information might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any variants.

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  1. ScreenIT

    SciScore for 10.1101/2021.09.21.461322: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: This study was approved by the Animal Research Committee of Saitama Medical University.
    IACUC: This study was approved by the Animal Research Committee of Saitama Medical University.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Peptide-pulsed cells were incubated for 3 hours at 37°C, and were stained with anti-HLA-A24 monoclonal antibody (mAb), A11.1M (44), followed by FITC-labeled goat anti-mouse IgG antibody (Sigma-Aldrich, St. Louis, MO).
    anti-HLA-A24
    suggested: None
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The resultant RMA-S-HHDA-24 cell line was cultured in RPMI-1640 medium (Nacalai Tesque Inc., Kyoto, Japan) with 10% FCS (Biowest, Nuaille, France) and 500 μg/ml G418 (Nacalai Tesque Inc.) Peptide binding assay: Binding affinity of each peptide to HLA-A*24:02 was measured by the peptide binding assay using RMA-S-HHD-A24 cells, as described before (42).
    RMA-S-HHDA-24
    suggested: None
    RMA-S-HHD-A24
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: We used HLA-A*24:02 transgenic mice which were kindly provided by Dr. François A. Lemonnier (Pasteur Institute, Paris, France).
    HLA-A*24:02
    suggested: RRID:IMSR_TAC:9663)
    Recombinant DNA
    SentencesResources
    HHD-A24 cDNA was subcloned into the mammalian expression plasmid, pcDNA3.1 (+) (Thermo Fisher Scientific, MA) (pcDNA3.1-HHD-A24).
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    The TAP2-dificient mouse lymphoma cell line, RMA-S (H-2b) was transfected with pcDNA3.1-HHD-A24 by electroporation (Gene Pulser Xcell, Bio-Rad, Hercules, CA), and cloned by the FACSAria II cell sorter (BD Biosciences, Franklin Lakes, NJ).
    pcDNA3.1-HHD-A24
    suggested: None
    Software and Algorithms
    SentencesResources
    Mean fluorescence intensity (MFI) of HLA-A*24:02 expression on the surface of RMA-S-HHD-A24 cells was measured by flow cytometry (FACSCanto II, BD Biosciences), and standardized as the percent cell surface expression by the following formula: % relative binding = [((MFI of cells pulsed with each peptide) – (MFI of cells incubated at 37°C without a peptide))/((MFI of cells incubated at 26°C without a peptide) – (MFI of cells incubated at 37°C without a peptide))] × 100.
    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: Thank you for sharing your data.

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.09.21.461322 on bioRxiv