T cell immunity might play a crucial role in long-term protection from severe COVID-19, even in the context of emerging VoC [16]. Therefore, it is essential to access cellular immunity with reliable and scalable assays, so that we can monitor more accurately the duration of cell-mediated immune protection against SARS-CoV-2 [16]. In the present study, we evaluated T cell responses 8–9 months after the immunization with 3 doses of BN162b2 mRNA vaccine with or without SARS-CoV-2 infection.
Compared to other cellular immunity assays, such as flow cytometry, ELISpot, and Next Generation Sequencing, QFN is less expensive, complex and requires minimal equipment [16].The assay consists of two antigen tubes, Ag1 and Ag2. The Ag1 tube contains epitopes derived from the S1 subunit of the Spike protein that stimulate production of IFN-γ from CD4+ T cells, while the Ag2 tube contains epitopes from the S1 and S2 subunits of the Spike protein that stimulate IFN-γ production from CD4+ and CD8+ T cells [8]. In QFN (+) participants, a positive response was detected more frequently in Ag2 tube than in Ag1 tube. In addition, Ag2 induced higher IFN-γ levels than Ag1, in participants with QFN (+). This finding is consistent with several other studies, which implies that CD4+ and CD8+ T cells contributed to the production of IFN-γ detected by the assay [8],[21],[22],[23],[24]. Martínez-Gallo et al found that the sensitivity of the assay ranged between 86–100%, 13 days after the second dose of BN162b2 mRNA vaccine [23].
In the study population, SARS-CoV-2 infection in unvaccinated children and adults did not stimulate IFN-γ production above the cut-off level of the assay. In accordance with these results, in a small pilot study, four out of five unvaccinated convalescent adults had negative IGRA prior to vaccination [23]. On the contrary, Murugesan et al, estimated that the overall sensitivity of QFN in unvaccinated convalescent adults measured from April 2020 to April 2021 was 84.5%, but it declined to 79% 10 months after infection [25]. In a small feasibility study, elevated IFN-γ responses in three out of four convalescent uninfected subjects were detected using QFN [8]. However, the above studies were conducted before the emergence of the Omicron variant.
Omicron variant has a high number of mutations in the Spike protein gene [14]. However, T cell epitopes are minimally affected, and the cell-mediated response is more resistant to VoCs than antibody response [14],[26],[27]. However, QFN may need validation in patients infected with other SARS-CoV-2 variants, such as Omicron, as it has been only validated against variant B.1.1.7 (Alpha) variant [27]. A test with a different group of peptides would probably be more appropriate for the detection of cellular immunity induced by the Omicron variant. A key question is if the cut-off of QFN that is set at 0.15 IU/mL can detect the cellular response after SARS-CoV-2 infection or if a lower cut-off could be proposed.
Using QFN, only 50% of COVID-19 naive participants immunized with mRNA 6-10.5 months after immunization with three BN162b2 mRNA were detected positive for INF-γ. According to our results, a study by Busà et al, 3 weeks after the third BN162b2 mRNA vaccine, found that almost half of the participants had not developed a good T cell response after the third dose [11]. In this study, cellular immunity was measured with QFN assay and ELISpot [11].
In contrast, previous studies have shown a higher positivity of the method in vaccinated (with 2 doses of mRNA vaccines) uninfected individuals than in our study[8] ,[23],[28]. However, it is worth noting that these studies were conducted in a shorter time interval (1–2 months) after vaccination with two doses of mRNA vaccines than our study.
QFN results were positive in 56% of individuals with hybrid immunity. Interestingly, the percentage of QFN(+) results was not statistically significantly higher in the hybrid immunity versus the vaccinated group (50%) and there was no association between distance from vaccination or infection with QFN result in this group. On the contrary, higher IFN-γ levels, measured by QFN, have been observed in infected participants (with or without breakthrough infection) in other studies comparing the cellular response to vaccination (BNT162b2 mRNA vaccine) between uninfected and infected individuals [4],[29].
It is clear how breakthrough infection with Οmicron variant interferes with the results of QFN. In a study by Søraas A et al, breakthrough infections with Omicron and Delta variants resulted in similar levels of IFN-γ as measured by QFN, indicating conservation of T cell immunity [30].
In the present study, in almost half of the vaccinated adults (infected and uninfected) adults, the IFN-γ levels measured by QFN were not detectable 6-10.5 months after the third dose. All vaccinated participants had mount adequate humoral response to vaccination. This could be attributed to the waning of the cellular immune response against SARS-CoV-2 after a substantial postvaccination time interval and could underscore the importance of a booster dose.
A possible pathophysiological explanation for these results is the fact that IFN-γ secreted from specific T cells was not detected because of migration of SARS-CoV-2 specific memory T cells to lymphoid tissues, after a certain period of time [28, 31]. After their encounter with SARS-CoV-2 antigens, T cells circulate in the blood secreting IFN-γ [28]. Over time, specific T cells are located in lymph nodes, and regularly some of them circulate in the blood [28]. In the absence of antigenic stimuli, there is a possibility that the QFN cannot detect IFN-γ secreted from these specific T cells [28]. Thus, re-evaluating the threshold of the assay at a certain time point after vaccination should be considered.
A decline in cellular responses measured with QFN 6–7 months post-vaccination with two doses of BN162b2 mRNA vaccine has been observed in several studies [22],[24],[32][33]. Bonnet et al, found that specific cell mediated responces, measured by QFN, 6 months after vaccination, decreased by 41% in 91 participants and increased by 33% in 17 others [32]. In a cohort of 419 HCWs, after a median time of 7 months after second dose, 72.3% of the participants had a reactive response to at least one Ag of QFN assay. In the same study, an increasing time interval between vaccination and time of measurement was associated with declining cellular response as measured by QFN [22].
There are studies indicating that QFN may be less sensitive than other laboratory methods for T cell measurements. Tormo et al, reported that Flow Cytometry-Intracellular Staining assay (FC-ICS) demonstrated greater sensitivity in detecting T cell responses in vaccinated individuals with two doses of BN162b2 mRNA vaccine than the QFN assay [15]. These differences may be attributed partially to the different nature of the Spike antigens used in the assays, as QFN antigens may bind less to all Human Leukocyte Antigens (HLA-I and HLA-II) specificities compared to FC-ICS assay’s antigens [15]. Furthermore, Busà et al, observed that even though half of the participants did not develop a detectable with IGRA T cell effector response 3 weeks after the third dose with BN162b2 mRNA vaccine, all of them had specific Activated-Induced Marker (AIM) memory T cells, indicating the existence of memory T cells [11]. The AIM technique does not use HLA multimers for the detection of T cells and does not require prior knowledge of the type, epitope and cytokines of HLA to be interpreted [11],[16].
A limitation of our study is the limited number of participants included, especially in the unvaccinated group. Furthermore, our study does not estimate the kinetics of T cell and humoral responses at different time points. However, to our knowledge, there are not any previous published data with the QFN assay 6 months after vaccination, with three doses of the BNT162b2 mRNA vaccine.
In conclusion, SARS-CoV-2 QFN assay did not detect cellular immune responses in unvaccinated and a significant number of vaccinated or hybrid immunity individuals. The results of this assay, especially several months after immunization, must be interpreted with caution, as the clinical implications are not clear. Further comparative studies with different immunological assays are required to elucidate whether this is the result of low sensitivity of the assay or waning immunity to SARS-CoV-2.