A whole blood test to measure SARS-CoV-2-specific response in COVID-19 patients

Abstract

Objectives: To examine whether specific T-cell-responses to SARS-CoV-2 peptides can be detected in COVID-19 using a whole-blood experimental setting, which may be further explored as a potential diagnostic tool.

Methods: We evaluated interferon (IFN)-γ levels after stimulating whole-blood with spike and remainder-antigens peptides megapools (MP) derived from SARS-CoV-2 sequences; interleukin (IL)-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, eotaxin, basic fibroblast growth factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, Interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, Platelet-derived growth factor (PDGF), RANTES (regulated on activation, normal T cell expressed and secreted), tumour necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF) were also evaluated.

Results: IFN-γ-response to spike and remainder-antigens MPs was significantly increased in 35 COVID-19 patients compared with 29 'no COVID-19' individuals (medians spike-MP: 0.26 vs 0, p = 0.0002; medians remainder-antigens-MP: 0.07 vs 0.02; p = 0.02). This response was detected independently of patients' clinical parameters. IFN-γ-response to SARS-CoV-2-unrelated antigens cytomegalovirus (CMV) and Staphylococcal Enterotoxin B (SEB) was similar in COVID-19 compared with 'no COVID-19' individuals (median CMV: 3.46 vs 5.28, p = 0.16; median SEB: 12.68 vs 15.05; p = 0.1). In response to spike-MPs in COVID-19- compared with 'no COVID-19' -individuals, we found significant higher median of IL-2 (50.08 vs 0, p = 0.0018), IFN-γ (90.16 vs 0, p = 0.01), IL-4 (0.52 vs 0, p = 0.03), IL-13 (0.84 vs 0, p = 0.007) and MCP-1 (4602 vs 359.2, p = 0.05).

Conclusions: Immune response to SARS-CoV-2 peptides in a whole-blood assay is associated with COVID-19 and it is characterized by both Th1 and Th2 profile. This experimental approach may be useful for developing new T-cell based diagnostic tests for disease and vaccine settings.

Keywords: COVID-19; IFN-γ; Immune response; Multiplex analysis; SARS-CoV-2; Serology response; Specific response; T-cell based tests; Whole blood.

Graphical abstract

Fig. 1

Increased interferon (IFN)-γ response to SARS-CoV-2 peptides is associated with COVID-19. IFN-γ levels are significantly increased in COVID-19 patients compared with ‘no COVID-19’ patients after stimulating whole-blood with spike- (a) or remainder-antigens- megapools (MPs) (b). IFN-γ levels in response to the unrelated antigens CMV (c) and SEB (d) are similar in COVID-19 and ‘no COVID-19’ groups. 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 when ≤0.05. Blue dots highlight healthy donors. CD, cluster of differentiation; CMV, cytomegalovirus; SEB, staphylococcal enterotoxin B.

Fig. 2

The interferon (IFN)-γ response to SARS-Cov-2 peptides can be detected independently of the disease severity and symptoms onset. Evaluation of the IFN-γ response to spike- (a) and to remainder-antigens-megapools (MPs) (b) in COVID-19 patients according to the severity of the disease. No significant differences in the IFN-γ levels were found comparing patients with mild or moderate or severe/critical illness. Evaluation of the IFN-γ response to spike- (c) and to remainder-antigens- MPs (d) in COVID-19 patients according to symptoms onset. No significant differences were found in the IFN-γ levels stratifying patients based on symptoms onset in respect to IFN-γ testing. 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 ≤ 0.016 was considered significant.

Fig. 3

The interferon (IFN)-γ response to SARS-Cov-2 peptides can be detected independently on the number of lymphocytes. No significant differences were found in the IFN-γ levels in response spike- (a) and to remainder-antigens- megapools (MPs) (b) according to lymphocytes counts. The IFN-γ response to the Staphylococcal Enterotoxin B (SEB) (c) increases in patients with more than 1 × 10³/μL lymphocytes. Correlation of the lymphocyte counts and IFN-γ levels in response to spike- (d), remainder-antigens-MPs (e) and SEB (f); a significant (p = 0.05) but low positive correlation (r_s = 0.37) was found for SEB stimulation. IFN-γ was measured by ELISA in stimulated plasma. The horizontal lines represent the median; statistical analysis was performed using the Kruskall–Wallis test, Mann–Whitney test and Spearman's rank correlation test; p ≤ 0.05 or p ≤ 0.016 was considered significant.

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