Robust SARS-CoV-2-specific T cell immunity is maintained at 6 months following primary infection

Abstract

The immune response to SARS-CoV-2 is critical in controlling disease, but there is concern that waning immunity may predispose to reinfection. We analyzed the magnitude and phenotype of the SARS-CoV-2-specific T cell response in 100 donors at 6 months following infection. T cell responses were present by ELISPOT and/or intracellular cytokine staining analysis in all donors and characterized by predominant CD4⁺ T cell responses with strong interleukin (IL)-2 cytokine expression. Median T cell responses were 50% higher in donors who had experienced a symptomatic infection, indicating that the severity of primary infection establishes a 'set point' for cellular immunity. T cell responses to spike and nucleoprotein/membrane proteins were correlated with peak antibody levels. Furthermore, higher levels of nucleoprotein-specific T cells were associated with preservation of nucleoprotein-specific antibody level although no such correlation was observed in relation to spike-specific responses. In conclusion, our data are reassuring that functional SARS-CoV-2-specific T cell responses are retained at 6 months following infection.

Extended Data Figure 1. Set-up of cut-off point for ELISPOT responses against SARS-CoV-2 protein pools using pre-2020 healthy donors PBMC.

ELISPOT responses against SARS-CoV-2 protein pools from 17 pre-2020 healthy donors were performed using Spike (pools 1 and 2), N/M and Accessory proteins (ORF3a, ORF10, NSP8, NSP7A/b), with DMSO as negative control. Data in graph represented as SFC per million PBMC. Each point on violin plot represents a single donor. Bold black line represents median.

Extended Data Figure 2. Gating strategy for Intracellular cytokine staining.

First, lymphocytes were gated according to FS-A and SS-A before removing doublets according to FS-A and FS-H. Live T cells were then gated using an amine reactive red fixable viability dye and anti-CD3. Finally, CD4+ and CD8+ T cells were gated according to positive staining with anti-CD4 and anti-CD8, respectively.

Extended Data Figure 3. Characterisation of the Th cytokines released by SARS-CoV-2 specific cells after peptide stimulation.

Characterisation of the Th cytokines released by SARS-CoV-2 specific cells during peptide stimulation shows IL-2 is consistently the dominant cytokine released. Supernatant from the wells of ELISPOT assays from eleven responding donors was analysed to assess the release of cytokines representative of classical Th subsets. In addition to the shown, IL-5, -9, -13 and IL-17A/F were not detected. The significance was determined using RM Two-way ANOVA with Geisser-Greenhouse correction, Dunnett multiple comparisons test.

Figure 1. Robust T-cell immunity against SARS-CoV-2 is present in all donors at 6 months following primary infection.

A. ELISPOT responses against SARS-CoV-2 protein pools at 6 months following primary infection. Left panel: A representative ELISPOT from 1 of 95 donors against Spike (pools 1 and 2), N/M and Accessory proteins (ORF3a, ORF10, NSP8, NSP7A/b), with DMSO as negative control and CEFX peptide pools and anti-CD3 as positive controls. Right panel: Summary data of all patients (N=95) studied according to Spike, N/M and Accessory peptide pools. Data in graph represented as SFC per million PBMC. Each point on violin plot represents a single donor. Bold black line represents median. The significance between pools was determined using a Friedman test (two sided) with Dunn’s multiple comparison test, p < 0.0001(****). B. Aggregate ELISPOT response against SARS-CoV-2 proteins at 6 months following primary infection. The spot numbers were aggregated for individual donors and shown in a bar chart.

Figure 2. T-cell responses against SARS-CoV-2 are 50% higher in donors with an initial symptomatic infection.

The cohort was divided into two groups according to symptoms at initial infection. A. The aggregated T-cell response (as SFC per million PBMC) against all peptide pools was compared between patients with (N=52) and without (N=43) respiratory symptoms (P=0.0235). B. T-cell responses (as SFC per million PBMC) to Spike (pools 1 and 2) (P=0.0330), N/M (P=0.0330), and Accessory proteins (P=0.2647) were compared between patients with and without symptoms. Each point on violin plot represents a single donor. Bold black line represents median. The significance was determined using Mann-Whitney testing (two sided), p < 0.05(*).

Figure 3. Overall detection of SARS-CoV-2-specific T cell responses by ICS.

A. Proportion of donors (N=100) with a detectable IFN-γ and/or IL-2 response by ICS for CD4+ T cells and CD8+ T cells against Spike and Non-spike proteins 6 months following primary infection. B. Aggregated IFN-γ and IL-2 ICS responses for CD4⁺ and CD8⁺ T cells against Spike and Non-spike proteins (N=100). The significance was determined using Wilcoxon matched-pairs signed rank test (two sided), p < 0.0001(****). Error bars represent standard error of the mean.

Figure 4. SARS-CoV-2-specific T-cell responses are characterised by a predominant profile of IL-2 production.

A. Features of CD4+ and CD8+ T-cell responses against SARS-CoV-2 proteins by intracellular cytokine staining at 6 months. Representative flow plots of CD4+ (Top panel) and CD8+ (Bottom panel) T-cell responses against peptide pools from Spike or non-spike (aggregate of N, M, ORF3a, ORF10, NSP8 and NSP7A/b) proteins. B. Polyfunctional analysis of SARS-CoV-2-specific CD4+ and CD8+ T-cells at 6 months. Relative distribution of single or multiple cytokine responses in CD4+ (Top panel) and CD8+ (Bottom panel) T-cells, and pattern of co-expression of IL-2, IFN-γ, TNF and IL-4 in SARS-CoV-2-specific T-cells. C. Aggregate ICS responses for CD4+ and CD8+ T-cells against Spike and Non-spike proteins according to IFN-g and/or IL-2 production (N=100). The significance was determined using Wilcoxon matched-pairs signed rank test (two sided), p < 0.05(*), p < 0.01(**), p < 0.0001(****). The exact P values are: for CD4+ T cell and Spike vs Non-spike, IFNγ+/IL-2-p=0.6629, IFNγ-/IL-2+ p<0.0001 and IFNγ+/IL-2+ p=0.1536; for CD8+ T cells Spike vs Non-spike, IFNγ+/IL-2- p=0.0036, IFNγ-/IL-2+ p=0.3071 and IFNγ+/IL-2+ p=0.0336. Error bars represent standard error of the mean. D. Correlation of Spike and Non-spike responses according to IFN-γ and IL-2 production by CD4+ (left panel) and CD8+ (right panel) T-cells at 6 months (N=100). Spearman’s Rank correlation (two sided) was used to test the significance, and p value and r value (correlation coefficient) are indicated for each panel.

Figure 5. The magnitude of the T-cell response at six months correlates with peak antibody level.

A. Antibody levels against Spike (N=81), Nucleoprotein (N=94), and RBD (N=87) of all patients at each time point post infection were plotted. Each grey line represents an individual patient. The median antibody level over time is shown in red. B. The correlation of ELISPOT responses at 6 months against peak antibody levels (Spike: N=82, Nucleoprotein: N=94, and RBD: N=87) were assessed for each antibody. C. Correlation of ELISPOT responses at 6 months with rate of antibody decline (expressed as ratio of ‘antibody level at 2 months after peak level’: ‘antibody peak level’) (Spike: N=60, Nucleoprotein: N=79, and RBD: N=67). The line represents linear regression. Spearman’s Rank correlation (two sided) was used to test the significance, and p value and r value (correlation coefficient) are indicated in each panel.