Spike is the most recognized antigen in the whole-blood platform in both acute and convalescent COVID-19 patients

Abstract

Objectives: To identify the best experimental approach to detect a SARS-CoV-2-specific T cell response using a whole-blood platform.

Methods: Whole-blood from 56 COVID-19 and 23 "NO-COVID-19" individuals were stimulated overnight with different concentrations (0.1 or 1 μg/mL) of SARS-CoV-2 PepTivator® Peptide Pools, including spike (pool S), nucleocapsid (pool N), membrane (pool M), and a MegaPool (MP) of these three peptide pools. ELISA was used to analyse interferon (IFN)-γ levels.

Results: The IFN-γ-response to every SARS-CoV-2 peptide pool was significantly increased in COVID-19 patients compared with NO-COVID-19 individuals. Pool S and MegaPool were the most potent immunogenic stimuli (median: 0.51, IQR: 0.14-2.17; and median: 1.18, IQR: 0.27-4.72, respectively) compared with pools N and M (median: 0.22, IQR: 0.032-1.26; and median: 0.22, IQR: 0.01-0.71, respectively). The whole-blood test based on pool S and MegaPool showed a good sensitivity of 77% and a high specificity of 96%. The IFN-γ-response was mediated by both CD4⁺ and CD8⁺ T cells, and independently detected of clinical parameters in both hospitalized and recovered patients.

Conclusions: This easy-to-use assay for detecting SARS-CoV-2-specific T cell responses may be implemented in clinical laboratories as a powerful diagnostic tool.

Keywords: COVID-19; IFN-γ-release assay (IGRA); Membrane protein; Nucleocapsid protein; SARS-CoV-2; Spike protein; T cell response; Whole-blood.

Figure 1

Spike and MegaPool peptides were the most immunogenic SARS-CoV-2 antigens in the whole-blood platform. The IFN-γ level in response to SARS-CoV-2 peptides was significantly increased in COVID-19 patients compared with NO-COVID-19 individuals after stimulating whole-blood with 1 μg/mL of pool N (A) and 0.1 μg/mL of pool M (C), pool S (E) and MegaPool (G) antigens. The ROC analysis shows significant AUC results for pool N (AUC = 0.81, p < 0.0001, B), pool M (AUC = 0.74, p = 0.0008, D), but mainly for pool S (AUC = 0.90, p < 0.0001, F) and MegaPool (AUC = 0.89, p < 0.0001, H) antigens. For scoring purposes, the cut-off of 0.13 IU/mL and of 0.19 IU/mL were chosen for pools N and M antigens, respectively, that led to 61% sensitivity/96% specificity for pool N and 52% sensitivity/91% specificity for pool M. For scoring purposes, the cut-off of 0.13 IU/mL and of 0.24 IU/mL were chosen for pool S and MegaPool antigens, respectively, that led to 77% sensitivity/96% specificity for both antigens. IFN-γ was measured by ELISA in stimulated plasma. The horizontal lines represent the median; statistical analysis was performed using the Mann–Whitney test and ROC analysis, and p-value was considered significant if ≤0.05. IFN, interferon; COVID-19: coronavirus disease 19; N: nucleocapsid; M: membrane; S: spike; ROC: receiver operating curve.

Figure 2

The Sars-CoV-2-specific response was mediated by both CD4⁺ and CD8⁺ T cells. Freshly isolated PBMCs were stimulated overnight with different stimuli to evaluate by flow cytometry the CD4⁺ or CD8⁺ T cell-specific IFN-γ response. A) Percentage of CD4⁺ or CD8⁺ T cells producing IFN-γ in response to the positive control SEB. B) Percentage of IFN-γ⁺ CD4⁺ T cells in response to pools S, N and M. C) Percentage of IFN-γ⁺ CD8⁺ T cells in response to pools S, N and M. In all graphs the IFN-γ values were subtracted from the unstimulated control. The horizontal lines represent the median and statistical analysis was performed using the Friedman test and p ≤ 0.05 was considered significant. Footnotes: IFN, interferon; N, nucleocapsid; M, membrane; S, spike.

Figure 3

The IFN-γ response to pools N, M, S and MegaPool was detectable in both recovered and hospitalized COVID-19 patients. Analysis of the IFN-γ response to pools N (A), M (B), S (C) and MegaPool (D) antigens in COVID-19 patients either hospitalized (n = 47) or recovered (n = 9). No significant differences were observed in response to all stimuli between the two groups of COVID-19 patients. IFN-γ was measured by ELISA in stimulated plasma. Statistical analysis was performed using the Mann-Whitney test and p ≤ 0.05 was considered significant. IFN, interferon; COVID-19: coronavirus disease 19; N, nucleocapsid; M, membrane; S, spike.

Figure 4

The IFN-γ response to pool S and MegaPool peptides can be detected in COVID-19 patients with a negative IgG/IgM serology. Evaluation of the IFN-γ response to pools N (A–B), M (C–D), S (E–F) and MegaPool (G–H) antigens in hospitalized COVID-19 patients (n = 41) stratified according to the IgG and IgM serology score and disease severity (A, C, E, G) or symptom onset (B, D, F, H). The IFN-γ response to pool S and MegaPool antigens can be detected in COVID-19 patients with negative IgG and IgM serology. IFN-γ was measured by ELISA in stimulated plasma. The horizontal lines represent the median; statistical analysis was performed using the Mann–Whitney test, and p-value was considered significant if ≤0.008. IFN, interferon; N, nucleocapsid; M, membrane; S, spike.