Immunogenic SARS-CoV2 Epitopes Defined by Mass Spectrometry

  1. Ke Pan
  2. Yulun Chiu
  3. Eric Huang
  4. Michelle Chen
  5. Junmei Wang
  6. Ivy Lai
  7. Shailbala Singh
  8. Rebecca Shaw
  9. Michael MacCoss
  10. Cassian Yee

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Abstract

SARS-CoV-2 infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID19, the disease caused by SARS-CoV-2, T cell responses are important in mediating recovery and immune-protection. The identification of immunogenic epitopes that can elicit a meaningful T cell response can be elusive. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes; however, our previous studies find that ‘immunodominant’ SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing SARS-CoV-2. We postulated that immunogenic epitopes for SARS-CoV-2 are best defined by directly analyzing peptides eluted from the peptide-MHC complex and then validating immunogenicity empirically by determining if such peptides can elicit T cells recognizing SARS-CoV-2 antigen-expressing cells. Using a tandem mass spectrometry approach, we identified epitopes of SARS-CoV-2 derived not only from structural but also non-structural genes in regions highly conserved among SARS-CoV-2 strains including recently recognized variants. We report here, for the first time, several novel SARS-CoV-2 epitopes from membrane glycol-protein (MGP) and non-structure protein-13 (NSP13) defined by mass-spectrometric analysis of MHC-eluted peptides, provide empiric evidence for their immunogenicity to induce T cell response.

Significance Statement

Current state of the art uses putative epitope peptides based on in silico prediction algorithms to evaluate the T cell response among COVID-19 patients. However, none of these peptides have been tested for immunogenicity, i.e. the ability to elicit a T cell response capable of recognizing endogenously presented peptide. In this study, we used MHC immune-precipitation, acid elution and tandem mass spectrometry to define the SARS-CoV-2 immunopeptidome for membrane glycol-protein and the non-structural protein. Furthermore, taking advantage of a highly robust endogenous T cell (ETC) workflow, we verify the immunogenicity of these MS-defined peptides by in vitro generation of MGP and NSP13 peptide-specific T cells and confirm T cell recognition of MGP or NSP13 endogenously expressing cell lines.

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  1. ScreenIT

    SciScore for 10.1101/2021.07.20.453160: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    HLA class I molecules from the cleared lysate were incubated with anti-HLA-A, B, C monoclonal antibody (W6/32) coupled Sepharose-4B resin (GE Healthcare, IL, USA) at room temperature for 2 hours.
    anti-HLA-A
    suggested: None
    Sorting and expansion: After two stimulation cycles, an aliquot of each well was stained with custom PE-conjugated MHC tetramer folded with HLA matched SARS-CoV2 peptide, and with APC-Cy7 conjugated anti-CD8 antibody (Biolegend, CA, USA).
    anti-CD8
    suggested: None
    After expansion, the purity of antigen specific T cells were determined with anti-CD8 antibody and MGP-65, NSP13-448, NSP13-242, NSP13-134, or NSP13-400 tetramer staining again.
    NSP13-134
    suggested: None
    After washing, the cells were fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer Set (eBioscienceTM, NY, USA), and then stained with APC conjugated anti-CD137, FITC conjugated anti-CD69, PE conjugated anti-IFN-γ, Pacific blue conjugated anti-TNF-α antibody (all purchased from Biolegend, CA, USA).
    anti-CD137
    suggested: None
    anti-CD69
    suggested: None
    anti-IFN-γ
    suggested: None
    anti-TNF-α
    suggested: (BioLegend Cat# 430901, RRID:AB_2883995)
    Experimental Models: Cell Lines
    SentencesResources
    TAP-deficient T-B cell hybrid cell line T2, melanoma cell line A375 (HLA-A0101/0201), RPMI-7951 (HLA-A0101/0201) and package cell lines Phoenix-GP, 293T were purchased from ATCC (VA, USA).
    T2
    suggested: None
    Melanoma cell lines Hs-578T (HLA-A0301/2402) and M14 (HLA-A1101/2402) were purchased from NCI.
    Hs-578T
    suggested: None
    Melanoma cell line Mel624 (HLA-A0201) was the gift from Dr. Steven Rosenberg (NCI).
    Mel624
    suggested: RRID:CVCL_RA32)
    MGP-pLVX or NSP13-pLVX lentiviral vector were transfected into package cell line 293T, together with package vectors contain VSVG envelop vector to make lentivirus.
    293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    A375, Mel624, RPMI-7951, Hs-578T and M14 cell lines were infected with MGP-pLVX or NSP13-pLVX lentiviral vectors and the stable cell lines were screened with puromycin selection.
    M14
    suggested: None
    MGP-65, NSP13-448, NSP13-242, NSP13-134 or NSP13-400 specific T cells were added at effector-to-target (E:T) of 20:1 cell ratio for 4 hours.
    MGP-65
    suggested: None
    For MGP-65 specific T cell test, A375-MGP and Mel624-MGP cells labeled with 51Cr were used as ‘hot’ targets.
    Mel624-MGP
    suggested: None
    Similarly, for NSP13-448 specific T cells, A375-NSP13 and RPMI-7951-NSP13 cells labeled with 51Cr were used as hot targets.
    RPMI-7951-NSP13
    suggested: None
    Non-radiolabeled A375 cells pulsed with NSP13-448 peptide or VGLL1 HLA-A0101 peptide (LSELETPGKY) (36) were used as cold targets and control cold targets, respectively.
    A375
    suggested: None
    For NSP13-242 specific T cells, A375-NSP13 and Mel624-NSP13 cells labeled with 51Cr were used as hot targets.
    Mel624-NSP13
    suggested: None
    For NSP13-400 specific T cell test, Hs-578T-NSP13 and M14-NSP13 cells labeled with 51Cr were used as hot targets.
    M14-NSP13
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    A375-MGP, Mel624-MGP (HLA-A0201+, MGP+), A375-GFP, Mel624-GFP (HLA-A0201+, GFP+), were used to evaluate MGP-65 specific T cell activity; A375-NSP13, RPMI-7951-NSP13 (HLA-A0101+, MGP+), A375-GFP, RPMI-7951-GFP (HLA-A0101+, GFP+), for NSP13-448 specific T cell activity; A375-NSP13, Mel624-NSP13 (HLA-A0201+, NSP13+), A375-GFP, Mel624-GFP, for NSP13-242 specific T cell activity; Hs-578T-NSP13 (HLA-A0301+, HLA-A2402+, NSP13+), Hs-578T-GFP (HLA-A0301+, HLA-A2402+, GFP+), for NSP13-134 specific T cell activity and M14-NSP13 (HLA-A2402+, NSP13+), Hs-578T-NSP13, M14-GFP (HLA-A2402+, GFP+), Hs-578T-GFP, for NSP13-400 specific T cell activity.
    Mel624-GFP
    suggested: None
    Recombinant DNA
    SentencesResources
    Lentivirus transduction: The cDNA of membrane glyco-protein (MGP) and Non-structure protein 13 (NSP13) of ORF1b from SARS-CoV-2 were purchased from Genscript (NJ, USA) and cloned into lentiviral vector pLVX (TAKARA, CA, USA) with fusion of GFP.
    pLVX
    suggested: RRID:Addgene_101121)
    MGP-pLVX or NSP13-pLVX lentiviral vector were transfected into package cell line 293T, together with package vectors contain VSVG envelop vector to make lentivirus.
    NSP13-pLVX
    suggested: None
    Software and Algorithms
    SentencesResources
    Peptide selection and validation: To analyze the acquired MS/MS spectra, the spectra were searched against the Swiss-Prot protein database (version 2020_05) by using Mascot search engine node (version 2.6) within Proteome Discoverer 2.3.
    Mascot
    suggested: (Mascot, RRID:SCR_014322)
    The False Discovery Rate (FDR) was determined using “Target Decoy PSM Validator” node within Proteome Discoverer (version 2.3) and protein/peptide with an FDR of ≤ 1% being retained for further analysis.
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    Statistical analysis: Data analysis was performed using GraphPad Prism version 7.03.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Flow Cytometry data were analyzed using FlowJo (version 10).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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.

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  2. Version published to 10.1101/2021.07.20.453160 on bioRxiv