Accuracy of QuantiFERON SARS-CoV-2 research use only assay and characterization of the CD4+ and CD8+ T cell-SARS-CoV-2 response: comparison with a homemade interferon-γ release assay

Abstract

Objectives: In this study, we aimed to characterize the SARS-CoV-2-specific T cell response detected by the QuantiFERON SARS-CoV-2 research use only assay in terms of accuracy and T cell subsets involved compared with a homemade interferon (IFN)-γ release assay (IGRA).

Methods: We evaluated T cell response by the standardized QuantiFERON SARS-CoV-2 tubes (antigen [Ag]1 and Ag2) and a homemade IGRA quantifying IFN-γ response to SARS-CoV-2 spike peptides (homemade-IGRA-SPIKE test). We evaluated the T cell subsets mediating the specific response using flow cytometry.

Results: We prospectively enrolled 66 individuals: COVID-19 or post-COVID-19 subjects and NO-COVID-19-vaccinated subjects, including healthy donors and immunocompromised subjects. The standardized kit detected 62.1% (41/66) of T cell responders. Ag2 tube showed a higher IFN-γ quantitative and qualitative response. Ag1 tube response was mainly mediated by CD4⁺ T cells; Ag2 tube response was mediated by CD4⁺ and CD8⁺ T cells. The homemade-IGRA-SPIKE test detected a higher number of responders (52/66, 78.8%) than the QuantiFERON SARS-CoV-2 assay (P = 0.056). The response was found in both T cell subsets, although a higher magnitude and response rate was observed in the CD4⁺ T cell subset.

Conclusion: The QuantiFERON SARS-CoV-2 response is mediated by CD4⁺ and CD8⁺ T cells. A lower number of responders is found compared with the homemade-IGRA-SPIKE test, likely because of the different peptide composition.

Keywords: COVID-19; IFN-γ release assay (IGRA), T cell response; QuantiFERON SARS-CoV-2 tubes; Spike peptides; Whole-blood.

Figure 1

T cell response to QuantiFERON SARS-CoV-2 RUO tubes compared with homemade-IGRA-SPIKE test. Evaluation of the IFN-γ-specific T cell response using SARS-CoV-2 spike and Ag tubes in the enrolled population (n = 66) stratified as follows: (A) COVID-19 (n = 19), (B) post-COVID-19 (n = 7), (C) HDs (n = 13), (D) MS (n = 20) and (E) patients with IMID (n = 7). IFN-γ levels were assessed in plasma harvested from tubes (Ag1 and Ag2) or stimulated samples (spike) and reported by subtracting the background. Dashed lines represent the cut-offs (Ag1 and Ag2 tubes: 0.15 IU/ml; spike: 0.13 IU/ml). Black horizontal lines indicate medians. Black symbols indicate unvaccinated subjects, white symbols indicate vaccinated subjects, and red symbols indicate subjects with MS before a booster dose, as reported in the legend. The Kruskal-Wallis test adjusted with Dunn's multiple comparisons test was performed. A P <0.05 was considered significant. Ag = antigen; HDs = healthy donors; IFN = interferon; IGRA = interferon-gamma release assay; MS = multiple sclerosis; IMID = immune-mediated inflammatory diseases; RUO = research use only; N = number.

Figure 2

SARS-CoV-2-specific T cell response was detected by both QuantiFERON SARS-CoV-2 RUO and homemade-IGRA-SPIKE test. The enrolled subjects (n = 66) were stratified as follows: COVID-19 (n = 19), post-COVID-19 (n = 7), HDs (n = 13), MS (n = 20) and patients with IMID (n = 7). (A-C) Evaluation of the IFN-γ-specific T cell response using SARS-CoV-2 (A) Ag1, (B) Ag2, and (C) Mitogen tubes. (D) Evaluation of the IFN-γ-specific T cell response using the homemade-IGRA-SPIKE test based on whole-blood stimulation with spike (0.1 µg/ml) or (E) SEB (200 ng/ml), used as a positive control. IFN-γ levels were assessed in plasma harvested from tubes or stimulated samples. Values were reported as stimulation index (signal of stimulated samples divided by negative control signal). Black triangles indicate unvaccinated subjects and white dots vaccinated subjects. Red dots indicate patients before booster dose within the MS cohort. The Kruskal-Wallis test adjusted with Dunn's multiple comparisons test was performed. A P <0.05 was considered significant. Ag = antigen; HDs = healthy donors; IFN = interferon; IGRA = interferon-gamma release assay; MS = multiple sclerosis; IMID = immune-mediated inflammatory diseases; RUO = research use only; SEB = staphylococcal enterotoxin B.

Figure 3

IFN-γ-intracellular T cell response in both assays is mediated by either CD4⁺ or CD8⁺ T cells. PBMCs from HDs (n = 6) and patients post-COVID-19 (n = 5) were stimulated using QuantiFERON SARS-CoV-2 RUO tubes and spike of the homemade-IGRA-SPIKE test, and the frequency of IFN-γ-specific T cells was evaluated by flow cytometry. (A) Frequency of CD4⁺ T cells in all subjects tested. (B) Frequency of CD8⁺ T cells in all subjects tested. Each dot represents an individual. Red dots indicate post-COVID-19 subjects. Black lines represent medians. Dashed lines indicate the threshold value set at 0.005% (i.e., the lower frequency of response observed among positive responders). White dots indicate HDs and red dots post-COVID-19 subjects, as reported in the legend. The Friedman test adjusted with Dunn's multiple comparisons test was performed to compare paired data of Ag tubes and spike. A P <0.05 was considered significant. IFN = interferon; Ag = antigen; HDs = healthy donors; IGRA = interferon-gamma release assay; MIT = mitogen; PBMCs = peripheral blood mononuclear cells; RUO = research use only; SEB = staphylococcal enterotoxin B; N = number.