Evaluation of circadian rhythm of SARS-CoV-2 interferon-gamma release assay (IGRA) in healthy vaccinated individuals: a case-series

Introduction

The study of the immune system, particularly the cell-mediated system, has become a topic of enormous interest over the years. Assessing the response of T lymphocytes to exposure to viral or bacterial antigens has been routine laboratory practice since the introduction of the so-called quantiferon test (1).

It appears that cellular immunity, probably more than the humoral response, has been found to be effective in preventing adverse disease progression (2). The main players in this type of reaction are T lymphocytes, which almost exclusively recognize peptide-like antigens via a system of receptors that interact with antigen presenting cells (APCs) (which present the previously processed antigen on their membrane).

Depending on the specific glycoproteins on their membrane, T lymphocytes are divided into T CD4+ (also called T helpers), which preferentially recognize antigens from vesicular compartments, and T CD8+ (also called cytotoxic T lymphocytes or killer lymphocytes or CTLs), which preferentially recognize antigens from the cytosolic compartment.

T cells not only recognize and kill virus-infected cells, but also reduce viral replication by actively supporting the immune response in infected patients. TCD4+ and TCD8+ cells achieve this through their cytotoxic activity, the recruitment and/or priming of other immune cells and the secretion of important cytokines such as interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and interleukin 2 (3). Similar to memory B lymphocytes, T lymphocytes are activated on first contact with the antigen and then react on second contact by proliferating very rapidly and simultaneously producing specific effector T cells and the specific memory cell clone for subsequent contacts (4).

Due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there was an urgent need to develop highly automated tests to assess cellular immune responses to the infection and to new vaccines against SARS-CoV-2. The IFN-γ release assay (IGRA) SARS-CoV-2 (Roche Diagnostics, Basel, Switzerland) combines in vitro stimulation of T cells with SARS-CoV-2 antigens (IGRA SARS-CoV-2 Cobas tubes) and an automated electrochemiluminescence immunoassay (ECLIA) for measuring IFN-γ to qualitatively determine the T cell-mediated immune response to SARS-CoV-2. The surface of one of the IGRA tubes is coated with over 180 different SARS-CoV-2 antigens, ensuring broad coverage of viral RNA and host HLA subtypes and high sensitivity to a wide range of viral sublines.

The aim of our study was to investigate whether and how a commercial IGRA may vary throughout the day after coronavirus disease 2019 (COVID-19) vaccination, to establish whether this new test may be influenced by circadian rhythm, so that specific time point for collecting blood would need to be recommended. We present this article in accordance with the AME Case Series reporting checklist (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-24-45/rc).

Case presentation

This study involved, on a voluntary basis, 4 employees of the Pederzoli Hospital in Peschiera del Garda (median age: 31 years; 50% females) who had previously received a primary vaccination cycle, a single homologous booster vaccination with the monovalent mRNA vaccine BNT162b2 from Pfizer/Biontech and a single booster with the new bivalent mRNA vaccine BNT162b2 from Pfizer/Biontech (Comirnaty, Pfizer Inc, NY, USA) in November 2022.

Blood samples were collected more than three months after dose the administration of the last dose of the bivalent mRNA vaccine BNT162b2. On three consecutive days, a venous blood sample was collected from each subject at 9 am (T1), 2 pm (T2) and 5 pm (T3). Samples were collected using IGRA SARS-CoV-2 tubes (Roche Diagnostics, Basel, Switzerland) as follows: negative control (NC; a internal to establish baseline levels of interferon gamma in each individual sample set), positive control (PC; to determine T cell fitness and ensure the reliability of the response measured in the three sample sets) and Ag (antigen; to elicit the SARS-CoV-2 specific interferon gamma response).

More specifically, the PC test tube in the kit contains a mitogen and stimulates T lymphocytes independently of the pathogen, while the NC test tube does not contain any antigens.

Incubation of whole blood at 37 ℃ for 20 hours allows the stimulation of T cells with specific peptides coated on blood tubes, leading to production of IFN-γ in patients previously exposed to the coated antigens.

After incubation, the three blood tubes were centrifuged at 1,500 ×g for 5 minutes and IFN-γ concentration in the supernatant of the three IGRA SARS-CoV-2 tubes was measured using the Roche Elecsys IGRA SARS-CoV-2 test. Samples were classified as “reactive” when the difference between the IFN-γ levels in the AG and NC IGRA SARS-CoV-2 tubes was >0.013 IU/mL.

On the last day, a further sample was taken from all subjects to exclude an ongoing viral infection during the study period, using the MAGLUMI 2019-nCoV IgM assay (Snibe, Shenzhen, China). In addition, total anti-SARS-CoV-2 antibodies were determined using the Roche Elecsys Anti-SARS-CoV-2 ECLIA on a Roche Elecsys e411 to determine humoral immune status. Results are expressed as mean and percentage difference.

In 3 subjects (75%), a previous diagnosis of SARS-CoV-2 infection could be officially established by routine nucleic acid detection test (NAAT) screening. None of the subjects had n-CoV IgM levels of more than 1.10 AU/mL (cut-off) on day 3, while all subjects had total anti-SARS-CoV-2 antibody levels >25,000 U/mL.

The results of this study are shown in Figure 1 and Table 1. When analyzing the difference between IFN-γ levels in the AG and NC IGRA SARS-CoV-2 tubes, all subjects had levels >0.013 IU/mL and were thus reactive in all samples collected, as shown in Table 1. The percentage difference between IFN-γ measured at different time points did not show a uniform trend, as a reduction of IFN-γ levels was observed in 3/4 subjects at T2, while the IFN-γ T3 value was lower than the T1 value in half of the subjects. Between T2 and T3 an increase was found in all subjects, though in one of these the value was lower than the imprecision of the assay declared by the manufacturer (i.e., 1.04% vs. 1.5–1.9%). The decrease was larger in the only subject (#4) who had never tested positive for a previous SARS-CoV-2 infection. Even after adjusting the AG for the PC value, no circadian trend could be observed. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This retrospective observational study was reviewed and approved by the Ethics Committee of the Provinces of Verona and Rovigo (No. 59COVIDCESC; November 8, 2021). Written informed consent was obtained from the patients for publication of this case series. A copy of the written consent is available for review by the editorial office of this journal.

Figure 1 Results of a SARS-CoV-2 IGRA in four healthy vaccinated volunteers, whose blood was collected at three different times of the day. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; IGRA, interferon gamma release assay.

Discussion

The COVID-19 pandemic has led to the rapid development of several novel vaccine platforms, including Pfizer/BioNTech’s mRNA-based vaccine BNT162b2. The mRNA vaccine formulations showed a high level of protection and stimulated robust innate and adaptive immune responses (5,6), fostering the generation of neutralizing antibodies, whose circulating levels tend to decrease after some months post-vaccination (7). In contrast, analyses of the magnitude and durability of SARS-CoV-2-specific T cell responses are limited.

The combined magnitude of SARS-CoV-2-specific IFN-γ and T-cell responses directed against SARS-CoV-2 structural proteins has been reported to be a better correlate of protection against COVID-19 infection than antibody responses (8).

In a previous study (9), we used the novel ROCHE IGRA SARS-CoV-2 assay to compare both humoral and cellular responses in a cohort of healthcare workers before and after receiving the fourth dose (booster) of Pfizer/BioNTech’s bivalent BNT162b2 mRNA vaccine (Comirnaty, Pfizer Inc., NY, USA); we then extended our study to the very early T-cell response in recipients of the bivalent BNT162b2 vaccine by monitoring the progressive change in IFN-γ release along with total anti-SARS-CoV-2 antibodies from day 1 to day 14 post-vaccination (10).

In this new study, we wanted to extend our work and investigate whether IFN-γ release varies throughout the day and whether the timing of sampling can influence the assessment of subjects’ immune response; several studies describe diurnal variations in various immune parameters such as lymphocyte proliferation, antigen presentation and cytokine gene expression (11). These cellular timers provide circadian fluctuations in various critical immune cell functions, including phagocytosis, cytokine production, cell transport, cell expression and immune response.

T and B lymphocytes constantly circulate between blood, lymph and lymphoid tissue to ensure adequate immune surveillance throughout the body and have been shown to be subject to circadian control. In human peripheral blood, the total number of lymphocytes and naïve CD4 and CD8 T lymphocytes follows a diurnal rhythm with a minimum during the day and a maximum during the night (12). Dendritic cells (DC), involved in the activation of adaptive immune response to infections or post-vaccination also express all components of endogenous molecular clock and exhibit circadian rhythms of gene expression (13). In humans, the secretion of interleukin-6 (IL-6) exhibits a biphasic circadian rhythm (14), and IFN-γ shows a daily rhythm that depends on the Per-2 gene (15).

Nevertheless, the currently available information on the cell-mediated response against SARS-CoV-2 is still limited, and a standard T-cell assay is also lacking.

In our study, we established a standard protocol for all samples collected to reduce possible pre-analytical variables and to assess whether the circadian rhythm to which the immune cells are subject could influence the test result.

The percentage difference between the different time points of each subject appears to be enormously higher (Table 1) than that stated in the manufacturer’s instructions for use (inter-assay imprecision: 1.5–1.9%).

All samples tested were reactive; taking samples at different times and on different days did not result in any deviations in the results of the qualitative test. No significant differences were found in the evaluation of the quantitative AG value either, neither in relation to the NC nor in relation to the PC.

Gao et al. found that the total spike-specific CD8+ T cell response in circulation elicited by the infection was lower compared to vaccination, but we found no significant differences between the response of subjects who previously tested positive for the infection and were vaccinated compared to subjects who were only vaccinated. As Gao et al. reported, BNT162b2 vaccination induces a T cell response exclusively to spike peptides, as the vaccine encodes only this protein. In contrast, SARS-CoV-2 infection triggers a response against the entire viral antigens. In addition, they observed a different spike-specific CD8+ T cell response after vaccination and infection with regard to the different epitopes (16,17). Presumably, the kit we used is not able to detect this difference, and this would explain the fact that our study shows no differences between subjects.

The greater decrease in the subject who never tested positive for a previous SARS-CoV-2 infection, could be explained by the fact that the N protein is the most immunogenic structural protein and that the IgG responses to the S protein are lower in SARS-CoV-2 (18).

Conclusions

In summary, circadian variation does not appear to have influence on the qualitative result of an IGRA assay in COVID-19-vaccinated subjects. Nevertheless, the large interindividual variability observed in this four-case series suggests that the timing of sampling should be standardized when multiple assessments or interindividual comparisons are needed. Further studies with larger number of subjects would also be advisable to confirm our preliminary findings.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the AME Case Series reporting checklist. Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-24-45/rc

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-24-45/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-24-45/coif). G.L. serves as the Editor-in-Chief of Journal of Laboratory and Precision Medicine. L.P. serves as an unpaid editorial board member of Journal of Laboratory and Precision Medicine from July 2024 to June 2026. The other author has no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This retrospective observational study was reviewed and approved by the Ethics Committee of the Provinces of Verona and Rovigo (No. 59COVIDCESC; November 8, 2021). Written informed consent was obtained from the patients for publication of this case series. A copy of the written consent is available for review by the editorial office of this journal.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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