Antibody Responses In Non-Severe SARS-CoV-2 Infections Are Driven By CD4+ T cells and Age

  1. Amelie E. Murrell
  2. Ewono Eyoh
  3. Jeffrey G. Shaffer
  4. Monika L. Dietrich
  5. Ivy V. Trinh
  6. Thomas J. Yockachonis
  7. Shuangyi Bai
  8. Crystal Y. Zheng
  9. Celia V. Mayne
  10. Sofia E. Cabrera
  11. Anyssa Aviles-Amaro
  12. Addison E. Stone
  13. Saraswatie Rambaran
  14. Sruti Chandra
  15. Debra H. Elliott
  16. Ashley R. Smira
  17. Sara N. Harris
  18. Katharine E. Olson
  19. Samantha J. Bilton
  20. Medea J. Gabriel
  21. Nicole D. Falgout
  22. Emily J. Engel
  23. Alisha D. Prystowsky
  24. Bo Ning
  25. Tony Hu
  26. Jay K. Kolls
  27. Samuel J. Landry
  28. Stacy S. Drury
  29. John S. Schieffelin
  30. Kevin J. Zwezdaryk
  31. James E. Robinson
  32. Bronwyn M. Gunn
  33. Elizabeth B. Norton

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Abstract

SARS-CoV-2 infection causes a spectrum of clinical outcomes and diverse memory responses. Population studies indicate that viral neutralizing antibody responses are protective, but do not always develop post-infection. Other antiviral antibody effector functions, T-cell responses, or immunity to seasonal coronaviruses (OC43, 229E) have been implicated but not defined in all ages. Here, we identify that children and adult subjects generate polyfunctional antibodies to the spike protein after asymptomatic infection or mild disease, with some subjects developing cellular responses without seroconversion. Diversity in immunity was explained by two clusters distinguished by CD4+ T-cell cytokines, age, and antibodies to seasonal coronaviruses. Post-vaccination neutralizing responses were predicted by specific post-infection immune measures, including IL-2, spike-IgA, OC43-IgG1, 229E-IgM. We confirm a key role for CD4+ T cell cytokines in functionality of anti-spike antibodies, and show that antibody diversity is impacted by age, Th/Th2 cytokine biases, and antibody isotypes to SARS-CoV-2 and seasonal coronaviruses.

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  1. ScreenIT

    SciScore for 10.1101/2022.04.22.22274032: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Subjects or households with suspected or confirmed SARS-CoV-2 infection were recruited from the Greater New Orleans community under Tulane Biomedical Institutional Review Board (federalwide assurance number FWA00002055, under study number 2020-585).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Determination of antigen-specific antibody reactivity by multiplexed Luminex analysis: Recombinant SARS-CoV-2 antigens (full-length spike, RBD, and N) and the recombinant spike protein from OC43, HKU1, 229E, and NL63 (Frederick National Laboratories) were coupled with MagPlex beads (Luminex) via sulfo-NHS coupling chemistry.
    antigen-specific
    suggested: None
    HKU1
    suggested: None
    The Spike protein ELISA for IgG antibodies has been validated by testing a standard set of positive and negative samples provided by NCI SeroNet staff.
    IgG
    suggested: None
    NK92 cells in complete alphaMEM culture medium were added at 5 × 104 cells/well in the presence of 4 µg/ml brefeldin A (Biolegend Cat# 420601), 5 µg/ml GolgiStop (BD Biosciences Cat# 554724) and 0.15µg of anti-CD107a antibody (Clone H4A3 PE-Cy7, Biolegend Cat# 328618) for 5 hours at 37°C.
    anti-CD107a
    suggested: (BioLegend Cat# 328618, RRID:AB_11147955)
    Antibody-dependent neutrophil phagocytosis (ADNP): Protocol was adapted from [72]
    Antibody-dependent neutrophil phagocytosis
    suggested: None
    Beads were washed with PBS containing 15 mM EDTA and stained with an FITC-conjugated anti-guinea pig C3 antibody (MP Biomedicals).
    anti-guinea pig C3
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Neutralization of SARS CoV-2 in Pseudovirus Assay: CHO cells were generated and stably expressed ACE2 by transfecting CHO cells with an ACE2 expression plasmid containing the blasticidin resistance gene.
    CHO
    suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
    CHO-ACE2 cells were similar in SARS CoV-2 susceptibility to the 293T/ACE2 cell line developed by Dr.
    CHO-ACE2
    suggested: None
    Virus neutralization was measured in CHO/ACE2 cells.
    CHO/ACE2
    suggested: None
    Pseudoviruses were produced by co-transfection of the four plasmids into 293T cells grown in T75 flasks with Fugene 6 as transfection reagent.
    293T
    suggested: None
    Unbound antibodies were removed by centrifugation before adding THP-1 cells at 2.5×104 cells/well.
    THP-1
    suggested: None
    Recombinant DNA
    SentencesResources
    RBD (aa321-535) was similarly expressed in the phCMV plasmid and purified on Streptactin X affinity columns.
    phCMV
    suggested: RRID:Addgene_15802)
    A DNA fragment encoding SARS CoV-2 N protein, including its natural leader sequence was generated by PCR of full-length N protein gene from a lentiviral N Protein expression vector (pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro, which was a gift from Nevan Krogan (Addgene plasmid # 141391 ; http://n2t.net/addgene:141391; RRID:Addgene_141391, [68]).
    detected: RRID:Addgene_141391)
    These included an expression plasmid for full-length spike protein of the Wuhan-1 strain containing the D614G amino acid chain (VRC7480.G614) [70], a pCMV ΔR8.2 lentivirus backbone plasmid (VRC5602) [71], the VRC5601 plasmid pHR’ CMV Luc containing the firefly luciferase reporter gene [71], and VRC9260 for TMPRSS2 expression.
    pCMV ΔR8.2 lentivirus
    suggested: None
    VRC5601
    suggested: None
    pHR’
    suggested: None
    Software and Algorithms
    SentencesResources
    Neutralization titers were defined as the serum dilution (ID50) at which relative luminescence units (RLU) were reduced by 50% compared to virus control wells after subtraction of background RLUs (determined by GraphPad Prism, version 9 for macOS, GraphPad Software, San Diego, California USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Spike Glycoprotein (stabilized) from SARS-Related Coronavirus 2, Wuhan-Hu-1 with C-Terminal Histidine Tag, Recombinant from Baculovirus), and SARS-CoV-2 specific mega pools at 0.2 μg/well including PepTivator SARS-CoV-2 Prot_S (Miltinyi - 130-126-700), SARS-CoV-2 Prot_M (130-126-702), SARS-CoV-2 Prot_N (130-126-699) in 96-well U bottom tissue culture plate (CytoOne CC7672-7596) in 200 μl RPMI-1640 with 10% FBS.
    PepTivator
    suggested: None
    ) GraphPad Prism (version 9.0.0, GraphPad Software, San Diego, CA), JMP (version 16.2.0, SAS Institute, Inc.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    , Cary, NC), and SAS (version 9.4, SAS Institute, Inc.
    SAS Institute
    suggested: (Statistical Analysis System, RRID:SCR_008567)

    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:
    Study limitations primarily involved using SARS-CoV-2 infection to differentiate subjects rather than pre-pandemic samples. In addition, the assays were limited to peripheral blood samples and not tissue-specific responses, which included only effector functions to spike protein and cytokine secretion instead of T-cell subset analyses. Detection of secreted cytokines allowed a greater number of cytokines to be evaluated but prevented confirmation of cells producing cytokines as would be observed intracellular stained cytokines for specific T-cell populations. However, cytokines between spike or peptide pools were highly correlated (Figure S5), indicating T-cell production. Also, high expression of IL-2 has been routinely observed from CD4+ T-cell and not CD8+ T-cells after SARS-CoV-2 infection [28, 38]. In this study, IL-17A secretion was closely correlated to IL-2 and Th1 cytokine release after stimulation with protein or peptide pools, suggesting that IL-17A may be serving as a proxy for a Th1/Th17 subset, as identified in other post-vaccination studies [61] which should be more closely examined. Finally, while the critical role for age in SARS-CoV-2 immunity was validated, it remains an ongoing question of why children exhibit less severity with infection and how differences in qualitative features of immunity depend on patient age. Our study used samples collected from subjects only shortly after the pandemic which will be difficult to perform as COVID subsides and vaccin...

    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 10.1101/2022.04.22.22274032v1 on medRxiv