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SARS-CoV-2 variants of concern partially escape humoral but not T-cell responses in COVID-19 convalescent donors and vaccinees

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Science Immunology  25 May 2021:
Vol. 6, Issue 59, eabj1750
DOI: 10.1126/sciimmunol.abj1750
  • Fig. 1 HCW study design.

    N=121 HCW were enrolled in a prospective SARS-CoV-2 infection and vaccination study. Upon symptomatic presentation to occupational health services a paired nasopharyngeal swab and EDTA blood sample was obtained (T0). A second EDTA blood sample was obtained 3 weeks after diagnostic RT-PCR (T3). Based on the diagnostic RT-PCR result at T0 and serology result at T3, 98 COVID-19 naive (yellow) and 23 COVID-19 recovered (blue) HCW were enrolled in the vaccination study on average 50 days after inclusion. N=13 COVID-19 recovered and N=12 COVID-19 naive participants were randomly selected for in-depth analysis. Blood samples were collected after the first (Vx13) and second (Vx23) vaccination, processed and subsequently used for downstream serological and cellular assays.

  • Fig. 2 Detection of SARS-CoV-2-specific humoral responses.

    Total immunoglobulin levels were measured in COVID-19 naive (yellow) and recovered (blue) donors at the acute, convalescent, post-vaccination 1 and post-vaccination 2 stage (T0, T3, Vx13, Vx23) by an (A) ELISA against nucleocapsid (N) and (B) receptor-binding domain (RBD). (C) Quantitative IgG against S1 was measured by a Luminex bead assay. (D) Antibody binding to WT SARS-CoV-2 and VOC B.1.1.7 and B.1.351 was determined by endpoint titration in ELISA. Virus neutralization was measured by PRNT50 against (E) WT SARS-CoV-2 (D614G) and (F) VOC. Analyses in panel B and C were performed on 121 participants, in-depth analyses in panel A, D, E, F on 25 participants. Timepoints in panel A, B and C were compared by performing a non-parametric repeated measures Friedman test. Endpoint titers between VOC in panel D were compared by RM one-way ANOVA or Friedman test. PRNT50 titers in panel D and E were compared by RM one-way ANOVA. * p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001. Symbol shapes indicate individual donors and are consistent throughout the figures. Lines in panel A and B show the mean, lines in panel C, D, E and F show geometric means. Dotted lines represent cut-off values for positivity (3x background OD450 in A, OD450 ratio = 1 in B, 10,08 BAU/ml in C). NT: not tested.

  • Fig. 3 Detection of ADCC-mediating antibodies by measuring NK92.05 degranulation.

    (A) Gating strategy for detection of degranulating NK cells: (1) NK92.05-CD16 cells are selected on basis of size and granularity, (2) exclusion of doublets, and (3) selection of LIVE and CD56+ cells. Degranulation is measured as percentage CD107a+ cells within the NK fraction, PBS coating is included as background control. (B-C) ADCC-mediating antibodies were detected in COVID-19 naive (yellow) and recovered (blue) donors at the acute, convalescent, post-vaccination 1 and post-vaccination 2 stage (T0, T3, Vx13, Vx23) against the WT N (B) and S (C) protein. (D) ADCC-mediating antibody reactivity with WT SARS-CoV-2 and VOC B.1.1.7 and B.1.351. These analyses were performed on 25 participants. Timepoints in panel B and C were compared by performing a non-parametric repeated measures Friedman test. Differences between variants were assessed by mixed-effect models. * p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001. Symbol shapes indicate individual donors and are consistent throughout the figures. Lines indicate mean responses.

  • Fig. 4 Detection of S-specific T cells by measuring up-regulation of activation-induced markers (AIM).

    (A) Gating strategy for virus-specific T cells cells that up-regulate AIM: (1) Lymphocytes are selected on basis of size and granularity, (2) exclusion of doublets, (3) selection of LIVE and CD3+ cells, and (4) division into CD4+ and CD8+ T cells. Activation is measured as percentage CD69+/CD137+ double-positive cells within the CD4 or CD8 fraction, DMSO stimulation is included as background control. (B, C, D) Antigen specific activation of CD4+ and CD8+ T cells in COVID-19 naive (yellow) and COVID-19 recovered (blue) donors at the acute, convalescent, post-vaccination 1 and post-vaccination 2 stage (T0, T3, Vx13, Vx23) by overlapping peptide pools covering the full WT S protein. Activation of SARS-CoV-2 specific CD4+ T cells is shown as percentage AIM+ cells within the CD4+ subset after (B) subtraction of the DMSO background or (C) as a stimulation index (SI) by dividing specific activation over background activation. (D) Activation of SARS-CoV-2-specific CD8+ T cells is shown as SI. An SI-index of 2 or higher is considered a positive T-cell response. (E-F) Antigen-specific activation of CD4+ T cells by peptide pools exclusively covering mutational regions in VOC B.1.1.7 and B.1.351, compared against homologous WT peptide pools. Antigen-specific T-cell responses are shown as SI. These analyses were performed in 20 participants. Timepoints in panel B, C and D were compared by performing a Kruskal-Wallis test. Differences between variants were compared by performing Wilcoxon test. * p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001. Symbol shapes indicate individual donors and are consistent throughout the figures. Lines indicate mean (B) or geometric mean (C, D, E, F) responses. Low cell count samples (<10,000 or <5,000 events within CD4+ or CD8+ gate, respectively) were excluded.

  • Table 1 Characteristics of study participants prior to vaccination.

    AllCOVID-19
    Recovered
    COVID-19
    Naive
    In-depth analysis
    COVID-19 RecoveredCOVID-19 Naive
    N12123981312
    Gender
    Male39 (32.2%)6 (26.1%)33 (35.6%)3 (23%)1 (8.3%)
    Female82 (67.8%)17 (73.9%)65 (66.3%)10 (77%)11 (91.7%)
    Age404238.54247
    (median + Q1-Q3)(34.8-55.8)(34.5-56.5)(34.3-57.3)(34.5-56.5)(34.3-55.0)
    < 3021 (17.4%)4 (17.4%)17 (17.3%)3 (23.1%)1 (8.3%)
    30-4456 (46.3%)10 (43.5%)46 (46.9%)5 (38.5%)5 (41.7%)
    45-5935 (28.9%)7 (30.4%)28 (28.6%)4 (30.8%)5 (41.7%)
    > 609 (7.4%)2 (8.7%)7 (7.1%)1 (7.7%)1 (8.3%)
    Days between diagnosis and vaccination (median)-54-47-
    < 30 days2 (8.7%)2 (15.4%)
    30-60 days12 (52.2%)10 (85.6%)
    >60 days9 (39.1%)1 (7.7%)

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/6/59/eabj1750/DC1

    Figure S1. Normalized S-curves for determining PRNT50

    Figure S2. Correlation between antibody binding and Fc-mediated functionality

    Figure S3. Detection of SARS-CoV-2-specific T cells using OX40

    Figure S4. Detection of S-specific T cells by measuring upregulation of activation-induced markers (AIM)

    Figure S5. Detection of S-specific T cells by measuring production of IFNγ in culture supernatant

    Table S1. Overview of immunological responders

    Table S2: Comparison of geometric mean S1-specific IgG BAU/ml (MIA) between in-depth analyzed sera and all sera

    Table S3. PRNT50 fold change reactivity to VOC

    Data file S1. Raw data file (Excel spreadsheet)

  • The PDF file includes:

    • Figs. S1 to S5
    • Tables S1 to S3

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    Other Supplementary Material for this manuscript includes the following:

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