Low antigen abundance limits efficient T-cell recognition of highly conserved regions of SARS-CoV-2

  1. Srividhya Swaminathan
  2. Katie E. Lineburg
  3. George R. Ambalathingal
  4. Pauline Crooks
  5. Emma J. Grant
  6. Sonali V Mohan
  7. Jyothy Raju
  8. Archana Panikkar
  9. Laetitia Le Texier
  10. Zheng Wei Marcus Tong
  11. Keng Yih Chew
  12. Michelle A. Neller
  13. Kirsty R. Short
  14. Harsha Gowda
  15. Stephanie Gras
  16. Rajiv Khanna
  17. Corey Smith

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Abstract

Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8 + T-cell epitopes across the nucleocapsid (N), spike (S) and ORF3a proteins of SARS-CoV-2 and five CD8 + T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8 + T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad pre-existing immunity in the community.

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  1. ScreenIT

    SciScore for 10.1101/2021.10.13.464181: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics approval to undertake the research was obtained from QIMR Berghofer Medical Research Institute Human Research Ethics Committee.
    Consent: Informed consent was obtained from all participants.
    Field Sample Permit: In-vitro expansion of SARS-CoV-2 specific T cells: PBMC were harvested from peripheral blood within 24 hours of collection.
    Sex as a biological variableParticipants ranged in age from 20 to 75, 24 were male and 36 were female, and were a median of 70 (46 – 131) days post-initial diagnosis.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    Conservation of ORF1ab SARS-CoV-2 isolates by variant: ORF1ab protein sequences for the SARS-CoV-2 (taxid ID 2697049, 419 amino acids) coronaviruses of the following Pango lineages: B1.1.7 (n=9505 sequences from 22.06.2021 until 16.07.2021, reported within as Alpha), B.1.351 (all available n=358 sequences, reported within as Beta), P.1 (all available n=6214 sequences, reported within as Gamma), B.1.617.2 (all available n=327 sequences, reported within as Delta), B.1.427 + B.1.429 (all available n=9850 sequences, reported within as Epsilon), P.2 (all available n=446 sequences, reported within as Zeta), B.1.525 (all available n=487 sequences, reported within as Eta), P.3 (all available n=5 sequences, reported within as Theta), B.1.526 (most recent n=9797 sequences from 07.05.2021 until 16.07.2021, reported within as Iota) and B.1.617.1 (all available n=116 sequences, reported within as Kappa) were obtained from the NCBI virus database (http://www.ncbi.nlm.nih,gov/labs/virus) on the 16th of July 2021.
    B1.1.7
    suggested: None
    Software and Algorithms
    SentencesResources
    In-silico identification of T-cell epitope determinants: The protein sequence of ORF1ab polyprotein (NCBI accession number: QXP49545.1) were retrieved using NCBI (https://www.ncbi.nlm.nih.gov) databases and saved in FASTA format for further analysis.
    NCBI
    suggested: (NCBI, RRID:SCR_006472)
    https://www.ncbi.nlm.nih.gov
    suggested: (GENSAT at NCBI - Gene Expression Nervous System Atlas, RRID:SCR_003923)
    Allele Frequency Net Database (http://www.allelefrequencies.net) was used as a platform to screen allele frequencies across various worldwide populations.
    http://www.allelefrequencies.net
    suggested: (Allele Frequencies in Worldwide Populations, RRID:SCR_007259)
    Later, the cells were washed and cultured in 48-well plates for 2 weeks supplemented with recombinant interleukin-2 (IL-2, Charles River Labs, USA) at 20 IU / mL every 2–3 days thereafter.
    Charles River Labs
    suggested: None
    Finally, cells were washed again and acquired using a BD LSRFortessa with FACSDiva software.
    FACSDiva
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    FlowJo LLC, Ashland, Oregon)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Proteomics analysis: Mass spectrometry raw files were downloaded from PRIDE (PXD018594, PXD017710) and MassIVE repository (MSV000085734) and searched against the UniProt Human protein database and RefSeq SARS-CoV-2 protein database using Sequest HT through Proteome Discoverer (Version 2.2) (Thermo Scientific, Bremen, Germany).
    PRIDE
    suggested: (Pride-asap, RRID:SCR_012052)
    RefSeq
    suggested: (RefSeq, RRID:SCR_003496)
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    Statistical analysis: GraphPad Prism 8.2.1 (San Diego, CA) was used to perform statistical analysis.
    GraphPad Prism
    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: We detected the following sentences addressing limitations in the study:
    While limitations in the availability of HLA-matched target cells did not allow us to perform endogenous antigen presentation assays with SARS-CoV-2 infected cells, our observations on antigen abundance from proteomic studies of SARS-CoV-2 infection do suggest that limited antigen abundance could impact T-cell priming against the conserved regions of ORF1ab. It is plausible that this may offer all coronaviruses an immune evasion strategy that limits recognition of highly conserved proteins that are critical for viral replication. More detailed analysis of antigen/epitope abundance and efficiency of T-cell recognition are required to determine this. One limitation of our study is the relatively homogenous population of COVID-19 convalescent volunteers used to assess T-cell responses. Limitations in the HLA diversity of our cohort likely contributed to the small number of epitopes we discovered. Observations in more genetically diverse individuals may reveal more epitopes. However, considering the devastating impact COVID-19 has had around the world, it seems unlikely that pre-existing cross-reactive CD8+ T cells induced by exposure to circulating coronaviruses are having a significant role in protection against severe disease, except in a small proportion of the population. Our observation suggests that the poor immunogenicity of critical regions of ORF1ab limits the efficiency of protection by pre-existing immunity.

    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

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  2. Version published to 10.1101/2021.10.13.464181 on bioRxiv