Genome-Wide Asymptomatic B-Cell, CD4 + and CD8 + T-Cell Epitopes, that are Highly Conserved Between Human and Animal Coronaviruses, Identified from SARS-CoV-2 as Immune Targets for Pre-Emptive Pan-Coronavirus Vaccines

  1. Swayam Prakash
  2. Ruchi Srivastava
  3. Pierre-Gregoire Coulon
  4. Nisha R. Dhanushkodi
  5. Aziz A. Chentoufi
  6. Delia F. Tifrea
  7. Robert A. Edwards
  8. Cesar J. Figueroa
  9. Sebastian D. Schubl
  10. Lanny Hsieh
  11. Michael J. Buchmeier
  12. Mohammed Bouziane
  13. Anthony B. Nesburn
  14. Baruch D. Kuppermann
  15. Lbachir BenMohamed

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Abstract

Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by emerging zoonotic CoVs: SARS-CoV, MERS-CoV, and the latest highly transmissible and deadly SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats, the natural hosts, and transmitted to humans via various intermediate animal reservoirs. Because there is currently no universal pan-Coronavirus vaccine available, two worst-case scenarios remain highly possible: (1) SARS-CoV-2 mutates and transforms into a seasonal “flu-like” global pandemic; and/or (2) Other global COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human population. Determining the antigen and epitope landscapes that are conserved among human and animal Coronaviruses as well as the repertoire, phenotype and function of B cells and CD4 + and CD8 + T cells that correlate with resistance seen in asymptomatic COVID-19 patients should inform in the development of pan-Coronavirus vaccines 1 . In the present study, using several immuno-informatics and sequence alignment approaches, we identified several human B-cell, CD4 + and CD8 + T cell epitopes that are highly conserved in: ( i ) greater than 81,000 SARS-CoV-2 human strains identified to date in 190 countries on six continents; ( ii ) six circulating CoVs that caused previous human outbreaks of the “Common Cold”; ( iii ) five SL-CoVs isolated from bats; ( iv ) five SL-CoV isolated from pangolins; ( v ) three SL-CoVs isolated from Civet Cats; and ( vi ) four MERS strains isolated from camels. Furthermore, we identified cross-reactive asymptomatic epitopes that: ( i ) recalled B cell, CD4 + and CD8 + T cell responses from both asymptomatic COVID-19 patients and healthy individuals who were never exposed to SARS-CoV-2; and ( ii ) induced strong B cell and T cell responses in “humanized” Human Leukocyte Antigen (HLA)-DR/HLA-A*02:01 double transgenic mice. The findings herein pave the way to develop a pre-emptive multi-epitope pan-Coronavirus vaccine to protect against past, current, and potential future outbreaks.

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    SciScore for 10.1101/2020.09.27.316018: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    The cells were subsequently washed with FACS buffer (1% BSA and 0.1% sodium azide in phosphate-buffered saline) and stained with anti-HLA-A2 specific monoclonal antibody (clone BB7.2) (BD-Pharmingen, San Diego, CA) at 4°C for 30 minutes.
    anti-HLA-A2
    suggested: None
    The following antibodies were used: CD8, CD4, CD62L, CD107a/b, CD44, CD69, TNF-α and IFN-γ).
    CD8
    suggested: (RayBiotech Cat# CS-11-0250, RRID:AB_1228199)
    CD4
    suggested: (RayBiotech Cat# CS-11-0133, RRID:AB_1228052)
    CD62L
    suggested: None
    CD44
    suggested: None
    CD69
    suggested: None
    TNF-α
    suggested: None
    Wells of 96-well Multiscreen HTS Plates (Millipore, Billerica, MA) were pre-wet with 70% methanol and then coated with 100 µl primary anti-IFN-γ antibody solution (10 µg/ml of 1-D1K coating antibody from Mabtech in PBS, pH 7.4, V-E4) OVN at 4°C.
    anti-IFN-γ
    suggested: None
    Plates were incubated at 4°C overnight with the sera, then washed with PBS-Tween 0.01% before to add anti-mouse IgG antibody (Mabtech – 1/500 dilution).
    anti-mouse IgG
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Immunization of mice: Groups of age-matched HLA transgenic mice/B6 mice (n = 3) were immunized subcutaneously, on days 0 and 14, with a mixture of four SARS-CoV-2-derived human CD4+ T/ CD8+T /B cell peptide epitopes delivered in alum and CpG1826 adjuvants.
    mice/B6
    suggested: None
    Sera of C57BL/6 mice immunized either with pool B cell peptides alum/CpG or adjuvant alone (control) were added into the wells at various dilutions (1/5, 1/25, 1/125 and 1/625 or PBS only, in triplicate).
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    The sequences were aligned using ClustalW algorithm in MEGA X.
    ClustalW
    suggested: (ClustalW, RRID:SCR_017277)
    The acquired data were analyzed with FlowJo software (BD Biosciences, San Jose, CA) and expression was measured by mean fluorescence intensity (MFI).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Constructing the Phylogenetic Tree: Phylogenetic analyses were conducted in MEGA X.
    MEGA
    suggested: (Mega BLAST, RRID:SCR_011920)
    Genome sequences of previous strains of SARS-CoV for human, bat, civet, and camel were retrieved from the NCBI GeneBank.
    NCBI GeneBank
    suggested: None
    Statistical differences observed in the measured CD8-, CD4-T cells and antibody responses between heatlhy donors and COVID-19 patients or across the different group of disease severity were calculated using ANOVA and multiple t-test comparison procedures in GraphPad Prism.
    GraphPad Prism
    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:
    • 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.

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  2. Version published to 10.1101/2020.09.27.316018 on bioRxiv