Comparative analysis between STANDARD‐E Covi‐FERON ELISA with pre‐existing IFN‐γ release assays and determination of the optimum cutoff value for assessment of T‐Cell response to SARS‐CoV‐2

Abstract Background Interferon‐gamma (IFN‐γ) release assays (IGRAs) are useful for the assessment of the T‐cell response to severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2). We aimed to assess the performance of the newly developed IGRA ELISA test compared to the pre‐existing assays and to validate the cutoff value in real‐world conditions. Methods We enrolled 219 participants and assessed agreement between STANDARD‐E Covi‐FERON ELISA with Quanti‐FERON SARS‐CoV‐2 (QFN SARS‐CoV‐2), as well as with T SPOT Discovery SARS‐CoV‐2 based on Cohen's kappa‐index. We further determined the optimal cutoff value for the Covi‐FERON ELISA according to the immune response to vaccinations or infections. Results We found a moderate agreement between Covi‐FERON ELISA and QFN SARS‐CoV‐2 before vaccination (kappa‐index = 0.71), whereas a weak agreement after the first (kappa‐index = 0.40) and second vaccinations (kappa‐index = 0.46). However, the analysis between Covi‐FERON ELISA and T SPOT assay demonstrated a strong agreement (kappa‐index >0.7). The cut‐off value of the OS (original spike) marker was 0.759 IU/mL with a sensitivity of 96.3% and specificity of 78.7%, and that of the variant spike (VS) marker was 0.663 IU/mL with a sensitivity and specificity of 77.8% and 80.6%, respectively. Conclusion The newly determined cut‐off value may provide an optimum value to minimize and prevent the occurrence of false‐negative or false‐positive during the assessment of T‐cell immune response using Covi‐FERON ELISA under real‐world conditions.


| INTRODUC TI ON
Coronavirus Disease 2019  continues to be a global concern despite various efforts that have been made to control its spread. Numerous extensive studies of the immune response to COVID-19 vaccination or to infections by SARS-CoV-2, which causes COVID-19, have elucidated the significance of T-cells in COVID-19 immunity. [1][2][3][4][5][6] One method for assessing the T-cell's response to SARS-CoV-2 is the interferon-gamma (IFNγ) release assay (IGRA), which is a blood-based test for diagnosing infectious diseases by measuring the IFNγ secreted by antigen-specific T-helper cells (Th cells) in blood samples. 7 The IGRA test was introduced in 2001 for the assessment of the IFNγ released by T-cells following stimulation by antigen-specific to Mycobacterium tuberculosis. 8 Following The Quanti-FERON (QFN) SARS-CoV-2 is a whole blood-based assay that can detect IFNγ produced by CD4+ and CD8+ T-cells in response to a SARS-CoV-2 peptide in the blood. 9 This assay is based on the same platform as the Quanti-FERON-TB Gold plus, which is a well-established diagnostic tool for latent tuberculosis infection. 10 By the end of 2021, the Quanti-FERON (QFN) SARS-CoV-2 has obtained a Conformite Europeenne (CE) mark and is deemed to meet EU safety, health, and environmental protection requirements; hence, it can be widely used to assess the T-cell response to  Meanwhile, the T SPOT Discovery SARS-CoV2 kit is a peripheral blood mononuclear cell (PBMCs)-based assay that allows the detection and enumeration of the SARS-CoV-2 specific T-cells. 12 Both types of IGRA tests have been used to evaluate the significance and durability of the T-cell response to SARS-CoV-2 infection or the COVID-19 vaccine in several published studies. 13,14 Nevertheless, although the IGRA kit showed potential as a T-cell evaluation tool, the sensitivity and accuracy of the IGRA assay remain an issue that requires further investigation. 15,16 The STANDARD-E Covi-FERON ELISA (RUO; SD BIOSENSOR), is a newly developed IGRA kit to evaluate the T-cell mediated immune response to SARS-CoV-2 specific proteins in heparinized whole blood. Although this kit is currently for research use only, studies on the T-cell response to SARS-CoV-2 using this kit demonstrated the potential of the STANDARD-E Covi-FERON ELISA as a useful tool for assessing T-cell response against SARS-CoV-2. 17,18 Hence, evaluation of whether the results from Covi-FERON ELISA are comparable to the results from pre-existing IGRAs is necessary.
Additionally, given that the sensitivity of the IGRA kit remains an issue, and that the sensitivity and specificity of the test are dependent on the cut-off value, it is crucial to validate whether or not the manufacturer's provided cut-off value is optimal in discriminating between positive or negative cases during the diagnostic test in the practical conditions. Therefore, in this study, we aimed to analyze the agreement between the newly developed IGRA kit, the STANDARD-E Covi-FERON ELISA, with two pre-existing IGRA assays, Quanti-FERON SARS-CoV-2, and T SPOT Discovery SARS-CoV-2 and determined the optimal cutoff value of STANDARD-E Covi-FERON ELISA according to the immune response that developed following SARS-CoV-2 infection and/or COVID-19 vaccination under real-world conditions. We also evaluated the performance of the STANDARD-E Covi-FERON ELISA when those newly determined cutoff values were applied.

| Study setting
This study was based on a longitudinal study design with 219 participants with a median age of 37. The enrolled participants comprised 41 COVID-19 patients and 178 healthy volunteers confirmed by real-time PCR tests who have fully received the COVID-19 vaccines.
The study period was from June 2021 to February 2022. People with a history of specific allergies, pregnant women, or someone receiving immunosuppressants were excluded from the study. All the participants provided written informed consent.

| Sample collection
Blood samples were collected at two time points from COVID-19 patients (before vaccination with at least 4 months after confirmed diagnosis and 4 weeks after vaccination with either BNT162b2 [Pfizer] or ChAdOx1-S [AstraZeneca]); and at three-time points from healthy volunteers (1 day before vaccination and 4 weeks following the first and second doses of ChAdOx1-S or BNT162b2 vaccines).

| Covi-FERON ELISA assay
The STANDARD-E Covi-FERON ELISA hereinafter refers to as Covi-FERON ELISA assay was performed according to the manufacturer's instruction. In brief, whole blood specimens were collected from

| Quanti-FERON SARS-CoV-2 assay
The Quanti-FERON SARS-CoV-2 assay hereinafter refers to as QFN SARS-CoV-2 was performed according to the manufacturer's instructions. Briefly, whole blood specimens were collected from the participants, and 1 mL was distributed into each of the QFN SARS-CoV-2 tubes (Nil tube [negative control], AG1 tube contains CD4 + epitopes derived from the receptor binding domain (RBD) that detect IFNγ production by CD4 + [AG1]; the AG2 tube contains CD4 + and CD8 + epitopes from S1 and S2 subunits that detect IFNγ production by CD4 + and CD8 + [AG2], and mitogen tube [positive control]). All tubes were incubated at 37°C for 24 h and then centrifuged at 2300 g for 15 min to harvest the plasma. The obtained plasma samples were subjected to ELISA to determine the amount of IFNγ.
The pre-determined cutoff value was ≥0.20 IU/mL. The OS and VS markers in the Covi-FERON ELISA and the AG1 and AG2 markers in the QFN SARS-CoV-2 were interpreted along with the other marker measurements from each kit to provide binary results for the presence or absence of the T-cell immune response to SARS-CoV-2.

| T SPOT Discovery SARS-CoV-2 assay
The T-SPOT Discovery SARS-CoV-2 assay, hereafter referred to as the T-SPOT assay, was performed according to the manufacturer's instructions. As described before, 19 the peripheral blood mononuclear cells (PBMCs) were isolated from 5 mL of whole blood samples and washed to remove any sources of interfering background signals.
Six wells were prepared for each sample: one nil control to identify non-specific cell activation, three wells to assess the SARS-CoV-2-

| Statistical analysis
We assessed the agreement between Covi-FERON ELISA and QFN SARS-CoV-2 or between Covi-FERON ELISA and T SPOT assay using Cohen's kappa-index and corresponding 95% confidence interval (95% CI). We determined the optimal cutoff value of the Covi-FERON ELISA markers to best discriminate the subjects with T-cell immune response and without response by building the general-    Table 3).

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We also determined the new cutoff value for Covi-FERON

| DISCUSS ION
The Covi-FERON ELISA is an IGRA kit that enables the detection of

CO N FLI C T O F I NTER E S T S TATEM ENT
No potential conflict of interest was reported by the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data from this study are available upon reasonable request.

I N S TITUTI O N A L R E V I E W B OA R D S TATE M E NT
This study was approved by the institutional review board (IRB) of Gyeongsang National University Changwon Hospital (IRB No. 2021-03-020).