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. 2021 Feb 5;371(6529):eabf4063.
doi: 10.1126/science.abf4063. Epub 2021 Jan 6.

Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection

Affiliations

Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection

Jennifer M Dan et al. Science. .

Abstract

Understanding immune memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for improving diagnostics and vaccines and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥6 months after infection. Immunoglobulin G (IgG) to the spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month after symptom onset. SARS-CoV-2-specific CD4+ T cells and CD8+ T cells declined with a half-life of 3 to 5 months. By studying antibody, memory B cell, CD4+ T cell, and CD8+ T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.

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Figures

Fig. 1
Fig. 1. Circulating antibodies to SARS-CoV-2 over time.
(A) Cross-sectional Spike IgG from COVID-19 subject plasma samples (n=228). Continuous decay preferred model for best fit curve, t1/2 = 140 days, 95% CI: 89-325 days. R = -0.23, p=0.0006. (B) Longitudinal Spike IgG (n=51), average t1/2 = 103 days, 95% CI: 65-235 days (C) Cross-sectional RBD IgG. Continuous decay preferred model for best fit curve, t1/2 = 83 days, 95% CI: 62 to 126 days. R = -0.36, p<0.0001. (D) Longitudinal RBD IgG, average t1/2 = 69 days, 95% CI: 58-87 days (E) Cross-sectional SARS-CoV-2 PSV neutralizing titers. One-phase decay (blue line) preferred model for best fit curve, initial t1/2 = 27 days, 95% CI 11-157d. R = -0.32. Continuous decay fit line shown as black line. (F) Longitudinal PSV neutralizing titers of SARS-CoV-2 infected subjects, average t1/2 = 90 days, 95% CI: 70-125 days. (G) Cross-sectional Nucleocapsid IgG. Continuous decay preferred model for best fit curve, t1/2 = 68 days, 95% CI: 50-106 days. R = -0.34, p<0.0001. (H) Longitudinal Nucleocapsid IgG, average t1/2 = 68 days, 95% CI: 55-90 days. (I) Cross-sectional Spike IgA titers. One-phase decay (blue line) preferred model for best fit curve, initial t1/2 = 11 days, 95% CI 5-25d. R = -0.30. Continuous decay fit shown as black line. (J) Longitudinal Spike IgA, t1/2 = 210 days, 95% CI 126-627 days. (K) Cross-sectional RBD IgA. One-phase decay (blue line) preferred model for best fit curve, initial t1/2 = 27 days, 95% CI: 15-59 days. R = -0.45. Continuous decay line fit shown in black. (L) Longitudinal RBD IgA, average t1/2 = 74 days, 95% CI: 56-107 days. For cross-sectional analyses, SARS-CoV-2 infected subjects (white circles, n=238) and unexposed subjects (gray circles, n=51). For longitudinal samples, SARS-CoV-2 subjects (n=51). The dotted black line indicates limit of detection (LOD). The dotted green line indicates limit of sensitivity (LOS) above uninfected controls. Unexposed = gray, COVID subjects = white. Log data analyzed in all cases. Thick blue line represents best fit curve. When two fit curves are shown, the thin black line represents the alternative fit curve.
Fig. 2
Fig. 2. Kinetics of SARS-CoV-2 memory B cell responses.
(A) Example flow cytometry plots showing staining patterns of SARS-CoV-2 antigen probes on memory B cells (See fig. S1 for gating). One unexposed donor and three convalescent COVID-19 subjects are shown. Numbers indicate percentages. (B) Gating strategies to define IgM+, IgG+, or IgA+ SARS-CoV-2 Spike-specific memory B cells. The same gating strategies were used for RBD- or Nucleocapsid-specific B cells. (C) Cross-sectional analysis of frequency (% of CD19+ CD20+ B cells) of SARS-CoV-2 S-specific total (IgG+, IgM+, or IgA+) memory B cells. Pseudo-first order kinetic model for best fit curve (R = 0.38). (D) Longitudinal analysis of SARS-CoV-2 Spike-specific memory B cells. (E) Cross-sectional analysis of SARS-CoV-2 RBD-specific total (IgG+, IgM+, or IgA+) memory B cells. Second order polynomial model for best fit curve (R = 0.46). (F) Longitudinal analysis of SARS-CoV-2 RBD-specific memory B cells. (G) Cross-sectional analysis of SARS-CoV-2 Nucleocapsid-specific total (IgG+, IgM+, or IgA+) memory B cells. Pseudo-first order kinetic model for best fit curve (R = 0.44). (H) Longitudinal analysis of IgG+ SARS-CoV-2 Nucleocapsid-specific memory B cells. (I) Cross-sectional analysis of SARS-CoV-2 Spike-specific IgG+ memory B cells. Pseudo-first order kinetic model for best fit curve (R = 0.49). (J) Longitudinal analysis of SARS-CoV-2 Spike-specific IgG+ memory B cells. (K) Cross-sectional analysis of SARS-CoV-2 Spike-specific IgA+ memory B cells. Second order polynomial model for best fit curve (|R| = 0.32). (L) Longitudinal analysis of SARS-CoV-2 Spike-specific IgA+ memory B cells. (M) Cross-sectional analysis of SARS-CoV-2 Spike-specific IgM+ memory B cells. Second order polynomial model for best fit curve (|R| = 0.41). (N) Longitudinal analysis of SARS-CoV-2 Spike-specific IgM+ memory B cells. (O) Fraction of SARS-CoV-2 antigen-specific memory B cells that belong to indicated Ig isotypes at 1-8 months PSO. Mean ± SEM. (P) Cross-sectional analysis of SARS-CoV-2 RBD-specific IgG+ memory B cells. Second order polynomial model for best fit curve (|R| = 0.51). (Q) Cross-sectional analysis of SARS-CoV-2 Nucleocapsid-specific IgG+ memory B cells. Second order polynomial model for best fit curve (|R| = 0.51). n = 20 unexposed subjects (gray circles) and n = 160 COVID-19 subjects (n = 197 data points, white circles) for cross-sectional analysis. n = 36 COVID-19 subjects (n = 73 data points, white circles) for longitudinal analysis. The dotted black line indicates limit of detection (LOD). The dotted green line indicates limit of sensitivity (LOS).
Fig. 3
Fig. 3. SARS-CoV-2 circulating memory CD8+ T cells.
(A) Representative flow cytometry plots of SARS-CoV-2-specific CD8+ T cells (CD69+ CD137+, See fig. S3 for gating) after overnight stimulation with S, N, M, ORF3a, or nsp3 peptide pools, compared to negative control (DMSO). (B) Cross-sectional analysis of frequency (% of CD8+ T cells) of total SARS-CoV-2-specific CD8+ T cells. Continuous decay preferred fit model, t1/2 = 125 days. R = -0.24, p = 0.0003. (C) Longitudinal analysis of total SARS-CoV-2-specific CD8+ T cells in paired samples. (D) Cross-sectional analysis of Spike-specific CD8+ T cells. Linear decay preferred model, t1/2 = 225 days. R = -0.18, p = 0.007. (E) Longitudinal analysis of Spike-specific CD8+ T cells in paired samples. (F, G) Distribution of central memory (TCM), effector memory (TEM), and terminally differentiated effector memory cells (TEMRA) among total SARS-CoV-2-specific CD8+ T cells. n = 169 COVID-19 subjects (n = 215 data points, white circles) for cross-sectional analysis. n = 37 COVID-19 subjects (n = 83 data points, white circles) for longitudinal analysis. The dotted black line indicates limit of detection (LOD). The dotted green line indicates limit of sensitivity (LOS).
Fig. 4
Fig. 4. SARS-CoV-2 circulating memory CD4+ T cells.
(A) Representative flow cytometry plots of SARS-CoV-2-specific CD4+ T cells (CD137+ OX40+, See fig. S4 for gating) after overnight stimulation with S, N, M, ORF3a, or nsp3 peptide pools, compared to negative control (DMSO). (B) Cross-sectional analysis of frequency (% of CD4+ T cells) of total SARS-CoV-2-specific CD4+ T cells. Continuous decay preferred fit model, t1/2 = 94 days. R = -0.29, p<0.0001. (C) Longitudinal analysis of total SARS-CoV-2-specific CD4+ T cells in paired samples from the same subjects. (D) Cross-sectional analysis of Spike-specific CD4+ T cells. Linear decay preferred model, t1/2 = 139 days. R = -0.26, p<0.0001. (E) Longitudinal analysis of Spike-specific CD4+ T cells in paired samples from the same subjects. (F, G) Distribution of central memory (TCM), effector memory (TEM), and terminally differentiated effector memory cells (TEMRA) among total SARS-CoV-2-specific CD4+ T cells. (H, I) Quantitation of SARS-CoV-2-specific circulating T follicular helper (cTFH) cells (surface CD40L+ OX40+, as % of CD4+ T cells. See fig. S5 for gating) after overnight stimulation with (H) Spike (S) or (I) MP_R peptide pools. (J) PD-1hi SARS-CoV-2-specific TFH at 1-2 months (mo) and 6 mo PSO. (K) CCR6+ SARS-CoV-2-specific cTFH in comparison to bulk cTFH cells in blood. For (A-E), n = 169 COVID-19 subjects (n = 215 data points, white circles) for cross-sectional analysis, n = 37 COVID-19 subjects (n = 83 data points, white circles) for longitudinal analysis. The dotted black line indicates limit of detection (LOD). The dotted green line indicates limit of sensitivity (LOS). For (H-J), n = 29 COVID-19 subject samples (white circles), n = 17 COVID-19 subjects at 1-2 mo, n = 12 COVID-19 subjects at 6 mo. The dotted black line indicates limit of detection (LOD). Statistics by (J) Mann-Whitney U test and (K) Wilcoxon signed-rank test. * p<0.05, **p<0.01, *** p<0.001.
Fig. 5
Fig. 5. Immune memory relationships.
(A) Relationship between gender and Spike IgG titers over time. Males: Linear decay preferred model, t1/2 = 110 days, 95% CI: 65-349 days, R = -0.27, p = 0.0046. Females: linear decay preferred model, t1/2 = 159 days, 95% CI 88-846 days, R = -0.22, p = 0.016. ANCOVA p = 0.00018. Test for homogeneity of regressions F = 1.51, p = 0.22. (B-E) Immune memory at 120+ days PSO in COVID-19 non-hospitalized and hospitalized subjects. Symbol colors represent peak disease severity (white: asymptomatic, gray: mild, blue: moderate, red: severe.) For subjects with multiple sample timepoints, only the final timepoint was used for these analyses. (B) Spike-specific IgG (left) and RBD-specific IgG (right) binding titers. n = 64 (non-hospitalized), n = 10 (hospitalized). Mann-Whitney U tests. (C) Frequency memory B cells specific to Spike (left) and RBD (right) at 120+ days PSO. n = 66 (non-hospitalized), n = 10 (hospitalized). Mann-Whitney U tests. (D) Frequency total SARS-CoV-2-specific CD8+ T cells (left) and Spike-specific CD8+ T cells (right). p = 0.72 for total SARS-2-CoV-specific, p = 0.60 for Spike-specific by Mann-Whitney U tests. n = 72 (non-hospitalized), n = 10 (hospitalized). (E) Frequency total SARS-CoV-2-specific CD4+ T cells (left) and Spike-specific CD4+ T cells (right). p = 0.23 for total SARS-CoV-2-specific, p = 0.24 for Spike-specific by Mann-Whitney U tests (F) Immune memory to SARS-CoV-2 during the early phase (1-2 mo, black line), medium phase (3-4 mo, red line), or late phase (5-8 mo, blue line). For each individual, a score of 1 was assigned for each response above LOS for RBD IgG, Spike IgA, RBD-specific memory B cells, SARS-CoV-2 specific CD4+ T cells, and SARS-CoV-2-specific CD8+ T cells, giving a maximum total of 5 components of SARS-CoV-2 immune memory. Only COVID-19 convalescent subjects with all five immunological parameters tested were included in the analysis. n = 78 (1-2 mo), n = 52 (3-4 mo), n = 44 (5-8 mo). (G) Percentage dot plots showing frequencies (normalized to 100%) of subjects with indicated immune memory components as described in (B) during the early (1-2 mo) or late (5-8 mo) phase. “G”, RBD-specific IgG. “B”, RBD-specific memory B cells. “4”, SARS-CoV-2 specific CD4+ T cells. “8”, SARS-CoV-2 specific CD8+ T cells. “A”, Spike-specific IgA. n = 78 (1-2 mo), n = 44 (5-8 mo). (H) Relationships between immune memory compartments in COVID-19 subjects over time, as ratios (full curves and data shown in fig. S10, B to F). AU = arbitrary units, scaled from fig. S10, B to F. “B/IgA”, RBD-specific memory B cell ratio to Spike IgA antibodies. “B/IgG”, RBD-specific memory B cell ratio to RBD IgG antibodies. “B/CD4”, RBD-specific memory B cell ratio to SARS-CoV-2-specific CD4+ T cells. “CD4/CD8”, SARS-CoV-2-specific CD4+ T cells ratio to SARS-CoV-2-specific CD8+ T cells. “CD4/IgG”, SARS-CoV-2-specific CD4+ T cells ratio to RBD IgG antibodies.

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