Highly functional Cellular Immunity in SARS-CoV-2 Non-Seroconvertors is associated with immune protection

  1. Athina Kilpeläinen
  2. Esther Jimenez-Moyano
  3. Oscar Blanch-Lombarte
  4. Dan Ouchi
  5. Ruth Peña
  6. Bibiana Quirant-Sanchez
  7. Anna Chamorro
  8. Ignacio Blanco
  9. Eva Martínez-Caceres
  10. Roger Paredes
  11. Lourdes Mateu
  12. Jorge Carrillo
  13. Julià Blanco
  14. Christian Brander
  15. Marta Massanella
  16. Bonaventura Clotet
  17. Julia G. Prado

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Abstract

The role of T cells in the control of SARS-CoV-2 infection has been underestimated in favor of neutralizing antibodies. However, cellular immunity is essential for long-term viral control and protection from disease severity. To understand T-cell immunity in the absence of antibody generation we focused on a group of SARS-CoV-2 Non-Seroconvertors (NSC) recovered from infection. We performed an immune comparative analysis of SARS-CoV-2 infected individuals stratified by the absence or presence of seroconversion and disease severity. We report high levels of total naïve and low effector CD8+ T cells in NSC. Moreover, polyfunctional Nucleocapsid (NP)-specific CD8+ T-cell responses, as well as reduced levels of T-cell activation monitored by PD-1 and activation-induced markers, were distinctive immunological traits in NSC. Longitudinal data support the stability of the NSC phenotype over three months. Our results implicate highly functional SARS-CoV-2 Spike and NP T-cell responses with low immune activation in protection from disease severity in the absence of seroconversion.

SUMMARY

To understand SARS-CoV-2 specific T-cell immunity in the absence of seroconversion, we characterized immunological features of Non-Seroconvertors recovered from infection. Highly functional specific T-cell responses and low immune activation were determinants of immune protection from severe disease.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.04.438781: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: All methods and experimental protocols of the study were approved by the Ethics Committee of Hospital Germans Trias i Pujol (PI-20-217).
    Consent: All the study subjects provided their written informed consent to participate.
    Sex as a biological variableFor the present study we identified participants (N=52) (median age 47 years, 62% female, 38% male) across Non-Seroconvertors (NSC, n=16), Low-neutralizers (LN, N=16) and Convalescent (Conv, N=15) (Fig 1A).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The LN were defined as having detectable anti-SARS-CoV-2 antibodies with neutralizing titers <500 IC50 values measured by pseudovirus neutralization assay.
    anti-SARS-CoV-2
    suggested: None
    Briefly, Nunc MaxiSorp ELISA plates were coated overnight at 4°C with 50 ml of capture antibody (anti-6xHis antibody, clone HIS.H8; ThermoFisher Scientific) at 2 mg/mL in PBS.
    anti-6xHis
    suggested: None
    The following reagents were used as secondary antibodies: HRP conjugated (Fab)2 Goat anti-human IgG (Fc specific) (1/20000), Goat anti-human IgM (1/10000), and Goat anti-human IgA (alpha chain specific) (1/20000) (all from Jackson Immunoresearch).
    anti-human IgG
    suggested: None
    anti-human IgM
    suggested: (LSBio (LifeSpan Cat# LS-C70420-10000, RRID:AB_10637387)
    anti-human IgA (alpha chain specific)
    suggested: None
    The second ELISA was a commercially available IgM and IgG class antibody ELISA against the SARS-CoV-2 NP (ImmunoDiagnostics, Hongkong).
    IgG
    suggested: None
    Anti-NP antibodies were captured by immobilized NP recombinant protein.
    Anti-NP
    suggested: None
    In brief, cells were labelled with a viability dye (APC-Cy7, Thermo Fisher Scientific) for 30 min at room temperature (RT) and surface stained for 30 min at RT with anti-human antibodies for CD3 (A700, clone UCHT1, BD), CD4 (FITC,clone OKT4, Biolegend), CD8 (V500, clone RPA-T8, BD), CD45RA (BV786, clone HI100, BD), CCR7 (PE-CF594, clone 150503, BD), CD27 (BV605, clone L128, BD), PD-1 (BV421, clone EH12.1, BD) and activation induced markers (AIM) CD25 (A647, clone BC96, Biolegend), OX40 (PE, clone Ber-ACT35, Biolegend) and CD137 (PeCy7 clone 41BB, Biolegend).
    APC-Cy7
    suggested: (Cell Signaling Technology Cat# 19805, RRID:AB_2798827)
    anti-human antibodies for CD3
    suggested: None
    CD4
    suggested: None
    CD8
    suggested: (BD Biosciences Cat# 560774, RRID:AB_1937325)
    CD45RA
    suggested: (Bio-Rad Cat# MCA88A647T, RRID:AB_1102080)
    BV786
    suggested: (BD Biosciences Cat# 563870, RRID:AB_2738459)
    CCR7
    suggested: (Bio-Rad Cat# MCA2367A647T, RRID:AB_2072907)
    CD27
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    PD-1
    suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106)
    BV421
    suggested: (BD Biosciences Cat# 743283, RRID:AB_2741401)
    CD25
    suggested: None
    A647
    suggested: (BioLegend Cat# 302617, RRID:AB_493046)
    CD137
    suggested: None
    PeCy7
    suggested: None
    Afterwards, cells were fixed with Fix/Perm Buffer A (Thermo Fisher Scientific) for 15 min at RT and stained intracellularly with Fix/Perm Buffer B and antibodies for TNF (PE-Cy7, clone MAb11, BioLegend), IFN-γ (BV711, clone B27, BD), and IL-2 (BV650, clone MQ1-17H12, BD) for 20 min at RT.
    PE-Cy7
    suggested: (BD Biosciences Cat# 560707, RRID:AB_1727542)
    IFN-γ
    suggested: (BD Biosciences Cat# 563416, RRID:AB_2738193)
    BV711
    suggested: (BD Biosciences Cat# 564039, RRID:AB_2738557)
    IL-2 (BV650
    suggested: (BioLegend Cat# 500334, RRID:AB_2563878)
    Experimental Models: Cell Lines
    SentencesResources
    For the neutralization assay, 200 TCID50 of pseudoviral supernatant was preincubated with serial dilutions of heat-inactivated serum or plasma samples (ranging from 1/60 to 1/14580) for 1h at 37°C and then added to ACE2-overexpressing HEK293T cells.
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Recombinant DNA
    SentencesResources
    Pseudovirus neutralization assay: HIV reporter pseudoviruses expressing SARS-CoV-2 S protein and Luciferase were generated. pNL4-3.
    pNL4-3
    suggested: None
    SARS-CoV-2.SctΔ19 was generated (Geneart) from the full SARS-CoV-2 S gene sequence with a deletion of the last 19 C-terminal codons (Ou et al., 2020), human-codon optimized and inserted into pcDNA3.4-TOPO.
    pcDNA3.4-TOPO
    suggested: None
    Expi293F cells were transfected using the Expifectamine Reagent (Thermo Fisher Scientific, Waltham, MA, USA) with pNL4-3.Luc.R-.E- and SARS-CoV-2.SctΔ19 at an 8:1 ratio, respectively.
    pNL4-3.Luc.R-
    suggested: None
    Software and Algorithms
    SentencesResources
    Study participants: The KING extension cohort is composed of 336 SARS-CoV-2 infected individuals, including 120 individuals who required hospitalization, 22% of whom with severe or critical SARS-CoV-2 infection (Fig 1A).
    KING
    suggested: (KING, RRID:SCR_009251)
    Finally, cells were resuspended and fixed in formaldehyde 1% and acquired on LSR Fortessa cytometer using FACSDiVa software (BD).
    FACSDiVa
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    Data analysis was performed using FlowJo software version 10.0.7 (Tree Star, Ashland, OR, USA).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analyses: Descriptive and comparison tests were performed using Graph Pad Prism, version 6 (GraphPad Software, Inc., San Diego, CA, USA).
    Graph Pad 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: We detected the following sentences addressing limitations in the study:
    Here, we overcome some of the previous studies limitations through a detailed identification of NSC and comparative analyses of total and S and NP-specific T cells. These data are essential to strengthen our understanding of T-cell mediated immunity and answer important questions regarding the identification of distinctive immunological traits associated with immune protection against SARS-CoV-2. We identified 4.8% of Non-seroconvertors in the KING extension cohort. We used a conservative approach to identify NSC by including confirmed SARS-CoV-2 PCR positivity, double ELISA testing and sampling-time estimation to allow for seroconversion. Longitudinal follow-up in the absence of SARS-CoV-2 IgG, IgM and IgA seroconversion over time further confirm the true nature of NSC in our study. Epidemiologically, NSCs were biased towards individuals with mild to moderate SARS-CoV-2 infection and a higher frequency of females. These data contrast with the presence of severe infection in 80% of Conv and higher frequency of males. The observed sex distribution is consistent with previous findings suggesting females exhibit robust T-cell responses and are less prone to severe SARS-CoV-2 infection and death than males (Takahashi et al., 2020; Jin et al., 2020; Vahidy et al., 2021). No significant differences in age distribution between NSC and Conv were found excluding age as one of the factors accounting for the differences in disease severity observed. We performed a characterization of T-...

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 41. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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 10.1101/2021.05.04.438781v1 on bioRxiv