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Rapid generation of durable B cell memory to SARS-CoV-2 spike and nucleocapsid proteins in COVID-19 and convalescence

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Science Immunology  22 Dec 2020:
Vol. 5, Issue 54, eabf8891
DOI: 10.1126/sciimmunol.abf8891
  • Fig. 1 Construct design and detection of SARS-CoV-2 RBD- and NCP-specific B cells

    (A) Recombinant spike receptor binding domain (RBD) and nucleocapsid protein (NCP) constructs of SARS-CoV-2. (B) SDS PAGE of purified, reduced (R) or non-reduced (NR) recombinant RBD and NCP. (C) Flow cytometry stainings of CD19+ B cells from an uninfected control and a recovered COVID-19 patient using double discrimination through inclusion of two fluorescent tetramers for each protein (RBD or NCP) in the same staining tube. Percentages indicate the proportions of RBD- or NCP-specific cells within total CD19+ B cells.

  • Fig. 2 Neutralizing antibodies and RBD-, and NCP- and HA-specific IgG antibody levels.

    (A) Neutralizing antibody titers to SARS-CoV-2 in 25 COVID-19 patients and 36 historic controls (sampled in 2019 and Q1 2020) as determined using a pseudovirus assay. Antigen-specific plasma IgG levels were determined to (B) SARS-CoV-2 RBD, (C) SARS-CoV-2 NCP and (D) influenza A/Michigan/2015 haemagglutinin (HA). Horizontal solid gray lines represent median values. (E) Neutralizing antibody titers, and IgG levels to (F) RBD and (G) NCP plotted against time since symptom onset of infection of 25 patients including 11 patients sampled twice. Patient datapoints are marked based on disease severity with severe as red triangles, moderate as orange diamonds and mild as blue circles. The 11 paired samples are connected with black lines. The dotted horizontal lines in panels A and E depict an ID50 of 20, the cut-off for neutralization (68). The dotted horizontal lines in B, C, F and G depict the cut-off for positivity, defined as +2SD of the controls. Statistics were performed with the Mann-Whitney U-test for unpaired data; **** p ≤ 0.0001.

  • Fig. 3 RBD- and NCP-specific Bmem cells predominantly express IgM or IgG1.

    (A) Gating strategy to discriminate T and B cells, followed by subsetting of total B cells into CD27-IgD+ naive, CD27+IgD+ Bmem and CD27+/−IgD- Bmem cells. Within IgD- Bmem cells, Ig switched subsets were defined based on the differential expression of IgG1, 2, 3, 4 subclasses and IgA. (B) Detection of RBD-specific (RBD+) B cells, and (C) NCP-specific (NCP+) B cells, utilized the same gating strategy as for total B cells. (D) Absolute numbers of IgG+ RBD+ and NCP+ Bmem cells in the first sample of 25 COVID-19 patients and 10 uninfected healthy controls. (E) Median frequencies of total, RBD+ and NCP+ Bmem subsets in 25 COVID-19 patients. Significant differences between RBD+ and NCP+ Bmem subsets are depicted with asterisks in the NCP column. (F) Frequencies of IgG+ Bmem cells expressing CD27 within total, RBD+ and NCP+ Bmem cells. Patient datapoints are marked based on disease severity with severe as red triangles, moderate as orange diamonds and mild as blue circles. Statistics: panel D, Mann-Whitney U-test for unpaired data; panels E and F, Wilcoxon matched-pairs signed rank test for paired samples; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

  • Fig. 4 Composition and kinetics of SARS-CoV-2 RBD- and NCP-specific Bmem in convalescence.

    Relative composition of the Bmem cell compartment based on Ig isotype and IgG subclass expression within (A) RBD-specific (RBD+) and (B) NCP-specific (NCP+) Bmem subsets. Patients’ data are ordered by days post-symptom onset. Absolute numbers of total, IgM+ and IgG+ Bmem cells specific for (C) RBD+ or (D) NCP+. Samples are plotted by days post-symptom onset for 25 individuals, with 11 patients sampled twice and paired samples connected with gray lines. Patient datapoints are marked based on disease severity with severe as red triangles, moderate as orange diamonds and mild as blue circles.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/54/eabf8891/DC1

    Figure S1. Absolute numbers of plasmablasts in COVID-19 patients

    Figure S2. Frequencies of RBD- and NCP-specific IgG+ Bmem cells expressing CD27

    Figure S3. Absolute numbers and frequencies of RBD- and NCP-specific Bmem cells expressing distinct Ig isotypes and IgG subclasses

    Figure S4. Absolute numbers of T helper cell subsets in COVID-19 patients

    Figure S5. Correlations between absolute numbers of RBD-specific Bmem and T helper cell subsets

    Figure S6. Correlations between absolute numbers of NCP-specific Bmem and T helper cell subsets

    Table S1. Patient characteristics

    Table S2. Immunological details of patients

    Table S3. Composition of the antibody panels

    Table S4. Antibody list

    Table S5. Flow cytometer set-up

    Table S6. Target values for 7th peak of rainbow beads in fluorescent channels

    Table S7. Raw data file (Excel spreadsheet)

  • The PDF file includes:

    • Fig. S1. Absolute numbers of plasmablasts in COVID-19 patients.
    • Fig. S2. Frequencies of RBD- and NCP-specific IgG+ Bmem cells expressing CD27.
    • Fig. S3. Absolute numbers and frequencies of RBD- and NCP-specific Bmem cells expressing distinct Ig isotypes and IgG subclasses.
    • Fig. S4. Absolute numbers of T helper cell subsets in COVID-19 patients.
    • Fig. S5. Correlations between absolute numbers of RBD-specific Bmem and T helper cell subsets.
    • Fig. S6. Correlations between absolute numbers of NCP-specific Bmem and T helper cell subsets.
    • Table S1. Patient characteristics.
    • Table S2. Immunological details of patients.
    • Table S3. Composition of the antibody panels.
    • Table S4. Antibody list.
    • Table S5. Flow cytometer set-up.
    • Table S6. Target values for 7th peak of rainbow beads in fluorescent channels.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S7. Raw data file (Excel spreadsheet).

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