QuantiFERON SARS-CoV-2 assay for the evaluation of cellular immunity after immunization with mRNA SARS-CoV-2 vaccines: A Systematic Review and Meta-Analysis

doi:10.21203/rs.3.rs-4308184/v1

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QuantiFERON SARS-CoV-2 assay for the evaluation of cellular immunity after immunization with mRNA SARS-CoV-2 vaccines: A Systematic Review and Meta-Analysis

https://doi.org/10.21203/rs.3.rs-4308184/v1

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A systematic review and meta-analysis was performed to evaluate the virus-specific T-cell response after COVID-19 mRNA vaccination, using the QuantiFERON-SARS-CoV-2 Interferon-γ Release Assay. A search was conducted (June 8, 2023) in the PUBMED, SCOPUS and medRxiv databases, to identify studies reporting the QuantiFERON-SARS-CoV-2 [Starter (two Antigen tubes) or Starter+Extended Pack (three Antigen tubes), cut-off³0.15IU/mL] Positivity Rate (PR) in immunocompetent adults, following the administration of two or three COVID-19 mRNA vaccine doses. Study quality was evaluated with the Critical Appraisal Skills Program Tool. A meta-analysis was conducted using a Random-Effects model. Heterogeneity and publication bias were assessed. Eleven eligible studies (with 5-75 vaccinated immunocompetent participants) were identified. For COVID-19-naive participants, £3 months after the second dose, the pooled PR (Random-Effects model) was 81 [95% Confidence Interval (95%CI):71-92]%. Comparing the Starter vs. the Starter+Extended Pack, a significant difference in PRs was detected (79.3% vs. 92.2%, p-value=0.039). At 3-6 and >6 months after the second dose and at ³3 months after the third dose, the pooled PRs were 59(95%CI:45-72)%, 79(95%CI:66-92)% and 66(95%CI:50-82)%, respectively. For convalescent participants, ³6 months after the third dose, the pooled PR was 81(95%CI:67-92)%. In conclusion, following the second or third COVID-19 mRNA vaccine, QuantiFERON-SARS-CoV-2 detected positive responses in a certain percentage of the vaccinees. This detection was higher when the Starter+Extended Pack was used. Possible explanations for the assay’s negative results in a subset of the participants could be: waning immunity, reduced sensitivity compared to other T-cell assays or lack of T-cell response induction in some vaccinees.

(PROSPERO Registration Number:CRD42023431315)

COVID-19

BNT162b2

mRNA-1273

T-cell

Interferon-γ Release Assay (IGRA)

QuantiFERON

The mRNA COVID-19 vaccines have considerably contributed to the mitigation of COVID-19 pandemic, as they have reduced SARS-COV-2-related morbidity and mortality [1]. COVID-19 mRNA vaccines use as a target the Spike (S) surface glycoprotein, which mediates the viral binding and entry through the Angiotensin-Converting Enzyme 2 (ACE2) receptor [1]. Immunization with these vaccines elicits a robust humoral response mediated mainly by neutralizing antibodies and a strong cellular response mediated by CD4⁺ and CD8⁺ T-cells [2, 3]. Although, neutralizing antibodies have been identified as correlates of vaccine-induced protection, an early and coordinated activation of both humoral and cellular immunity, is necessary for the virologic control and the limitation of COVID-19 severity after breakthrough infection [2, 4–6].

Previous studies describing the rapid antibody waning and the loss of antibodies’ neutralizing capacity against the emerging Variants of Concern (VoCs) have raised concerns regarding the protective efficacy of humoral immunity [7]. By contrast, T-cell responses largely retain their reactivity to VoCs as they can recognize a broader range of viral epitopes than neutralizing antibodies and many of their epitopes remain conserved across the VoCs [2, 8]. Evidence also suggest that T-cell responses induced after vaccination may have greater durability than antibody responses [6]. Notably, memory T-cell responses to the Nucleocapsid (N) protein were detected in patients 17 years after SARS-CoV infection [8, 9]. Additionally, increasing data support the critical role of T-cells in limiting the severity of COVID-19, as T-cells promote viral clearance by eradicating virus-infected cells and therefore control disease progression [10, 2, 11, 12]. For example, the early induction of SARS-CoV-2–specific Interferon-γ (IFN-γ)-secreting T-cells has been associated with a milder disease course, while severe clinical outcomes have been associated with the absence of these responses [2, 13].

As opposed to humoral responses, the role of SARS-CoV-2-spesific T-cell response in the host defense against the virus has been less extensively studied, owing to the technical complexity of T-cell measurement [14, 15]. Various methods have been employed in laboratories to determine the T-cell response elicited by SARS-CoV-2 infection or vaccination, with Flow Cytometry with Intracellular Cytokine Staining (FC-ICS) and ELISpot being the most widely used assays [16]. Enzyme-linked Immunosorbent Assay (ELISA)-IFN-γ Release Assays (IGRAs) consist of an alternative to these methods, as they are less time-consuming and complex, require minimal equipment and are simple to implement in the laboratory environment [17, 18].

QuantiFERON (QFN)-SARS-CoV-2 is a whole-blood based IGRA, that detects IFN-γ, released from CD4⁺ and CD8⁺ T-cells following stimulation with SARS-CoV-2 Antigens (Ags), with the use of ELISA [19]. The methodology in which QFN-SARS-CoV-2 is based, has been also applied in the clinical practice for the diagnosis of latent Tuberculosis and posttransplant Cytomegalovirus (CMV) infection [20, 21].

While several studies have used QFN-SARS-CοV-2 for the assessment of virus-specific vaccine induced T-cell response in different populations, a Systematic Review and Meta-Analysis of the application of this assay on immunocompetent adults has not yet been published. Therefore, the objective of the current study was the assessment of SARS-CoV-2-specific T-cell response post 2 or 3 doses COVID-19 mRNA vaccination using the QuantiFERON (QFN)-SARS-CoV-2 Interferon-γ Release Assay in immunocompetent adults.

We conducted a Systematic Review and Meta-Analysis to assess the Positivity Rate of QuantiFERON-SARS-CoV-2 in various time points after vaccination with mRNA vaccines, based on the Preferred Reporting Items of Systematic Review and Meta-Analysis (PRISMA) 2020 guidelines [22]. The protocol of the present study was registered in the International Prospective Register of Systematic Reviews (PROSPERO), under the registration number:CRD42023431315 [23].

Search strategy for identification of studies

PUBMED, SCOPUS and medRxiv databases were searched to identify relevant English Language studies published up to June 8, 2023. Three major groups of keywords were utilized on PUBMED: SARS-CoV-2, COVID-19, Severe Acute Respiratory Syndrome Coronavirus 2, Coronavirus disease 2019 (Group 1) AND QuantiFERON, IGRA, Interferon-gamma release assay, Interferon-γ release assay (Group 2) AND vaccin*, mRNA vaccine, BNT162B2, mRNA-1273 (Group 3). These three groups of keywords were combined with the word Boolean ‘AND’ and the terms within each category were combined with the Boolean ‘OR’. For the databases, SCOPUS and medRxiv, the keywords QuantiFERON and SARS-CoV-2, combined with the Boolean ‘AND’ were utilized.

Eligibility Criteria

To identify which studies were eligible for inclusion in the current Systematic Review and Meta-analysis, the PICOs (Population, Intervention, Comparator, Outcome, study design) framework was used. Full-text studies in English that used the QFN-SARS-CoV-2 ELISA and Starter Pack [two Antigen tubes: Antigen tube (Ag) 1, Ag2] or Starter plus Extended Pack (three Antigen tubes: Ag1,Ag2 plus Ag3) (Qiagen, Hilden, Germany) for the evaluation of the T-cell response after homologous immunization with the Moderna mRNA-1273 or the Pfizer-BioNTech BNT162b2 monovalent COVID-19 vaccine in immunocompetent adults (Population) and provided a qualitative result (Positivity Rate, n/N, %) of the assay (Outcome) were included in the present Systematic Review and Meta-Analysis. The included studies had to provide the time-point after immunization that the assay was carried-out (Outcome). In case both COVID-19 naïve adults and adults with hybrid immunity participated, the study had to provided separate QFN-SARS-CoV-2 Positivity Rates after immunization with COVID-19 mRNA vaccines, for the two groups, to be included (Outcome). The criteriums of Intervention and Comparator were not applicable in the current study. Cohort, case control and cross-sectional studies were suitable for inclusion (study design). The inclusion and exclusion criteria are presented in Supplementary Table 1.

Study Identification and Data Extraction

Following the assemblage of the studies from the databases, duplicate citations were excluded. The titles and abstracts of the remaining articles were screened by two independent investigators (M.M.D and A.M.) for topic relevance and eligibility. In case, insufficient information were provided in the title/abstract, the full text article was assessed to decide whether it should be included in the study. The reference lists of all relevant articles selected for inclusion were also screened to identify articles that met the predetermined criteria but were not retrieved in the initial electronic research.

A data extraction form was developed for this meta-analysis, collecting the following data: general study characteristics (author, setting, publication year, study design), study participants’ baseline characteristics (number of participants, age, gender, immune status, COVID-19 infection history, vaccine type and manufacturer, number of vaccines, time interval between last vaccination and QuantiFERON measurement), type of the QuantiFERON-SARS-CoV-2 assay used [Starter Pack (two Antigen Tubes: Ag1, Ag2) or Starter plus Extended Pack (3 Antigen Tubes: Ag1, Ag2 plus Ag3)] and the positivity rate of the QuantiFERON-SARS-CoV-2 assay at various time points after vaccination. Included studies with COVID-19 naïve participants were categorized based on the time interval of the measurement from vaccination, into the following groups: ≤3 months, 3–6 months, > 6 months from immunization after the second dose and ≥3 months after the third dose. Included studies with participants with hybrid immunity could be classified in only one group: ≥6 months from immunization after the third dose. In case a study provided measurements for two timepoints that belonged to the same category, the earlier measurements were used. The information provided from the data extraction were recorded in Microsoft Excel. Data extraction was performed by two independent researchers (M.M.D. and A.M.) and any discrepancies were resolved by a third researcher (G.K.).

Assessment of methodological quality

Two independent reviewers (M.M.D. and A.M.) assessed the quality of each study. Disagreements were discussed with a third researcher (G.K.). Reviewers were not blinded to study identifiers (e.g. author names, institutions, journals). The quality of studies was assessed using the Critical Appraisal Skills Program (CASP) Tool for cohort and case-control studies [24]. As there was no established cut-off point for the classification of studies as “good-quality studies” or “poor-quality studies”, we arbitrarily defined the threshold at 60%. This indicates that only studies fulfilling at least the 60% of criteria in each scale were considered of “good quality”.

Statistical Analysis

A meta-analysis was conducted using the command “metaprop” in STATA version 17.0 software (StataCorp, College Station, TX, USA), to estimate the pooled effect sizes with 95% Confidence Intervals (C.I.) for the Positivity Rate of QFN SARS-CoV-2. The results of meta-analysis are presented in forest plots. The pooled effect size was considered statistically significant when the number 0 was not included in the 95% CI. Heterogeneity among included studies was assessed using the I² statistic and Hedges Q statistic (with a p-value < 0.1 indicating statistically significant heterogeneity) [25]. An I² value ≥ 75% represented a high level of heterogeneity. In such cases, the Random-Effects model was applied to obtain the pooled effect sizes. Stratified analysis by region (type of QuantiFERON SARS-CoV-2 assay) was also conducted. In addition, leave-one-out sensitivity analysis was conducted, by removing one study each time, to identify the study that most influenced the results. Finally, Egger’s bias test and Funnel plot were used to assess potential publication bias [26].

Literature Search

The study selection PRISMA flow diagram is presented in Figure 1 [27]. Through the initial database search, 265 relevant studies were identified, from which 51 studies were removed as duplicates. From the remaining studies, 77 studies were considered irrelevant through title/abstract screening and 137 studies were assessed for eligibility through full text screening. According to the exclusion criteria, 125 studies were removed. From the remaining 12 studies, 10 studies met the inclusion criteria [28, 17, 29-36]. Regarding the two remaining studies, one article [37] was a letter to the Editor regarding the included study by Krüttgen et al, 2021 [31] and the other article [38] was an authors’ reply to this letter and provided additional information regarding the QFN-SARS-CoV-2 Positivity Rate for the aforementioned study and therefore was included in the meta-analysis. Finally, data regarding the use of QFN-SARS-CoV-2 assay from 11 studies were included in the present systematic review and meta-analysis [28, 17, 29-35, 38].

Characteristics and Quality Assessment of Included Studies

The main characteristics of the included studies are presented in Table 1. The studies were conducted in Spain [28], Greece [17, 34], Malaysia [29], Germany [31, 30], Bulgaria [32], Switzerland [36] and Belgium [35]. Regarding the study design, eight studies were cohorts [28, 17, 30, 31, 33-35]and two studies was case-controls [32, 36].

Α total of 1115 participants were enrolled in the above studies. The participants of the studies were not exclusively mRNA vaccinated immunocompetent adults, as some studies included also unvaccinated participants [17, 32], participants immunized against SARS-CoV-2 with other COVID-19 vaccines and immunocompromised patients [e.g. patients with chronic kidney disease including kidney transplant recipients [28], patients undergoing hemodialysis [28, 35] or peritoneal dialysis [28], patients under CD20 B-cell depleting therapy [36] and participants receiving anti-cancer treatments or mycophenolate mofetil [34]]. In the included studies, the number of immunocompetent mRNA-vaccinated participants whose SARS-CoV-2-specific T-cell response was evaluated with QFN-SARS-CoV-2 assay, varied ranging from 5-75 participants. In many included studies, these participants were Health Care Workers [30, 17, 32, 34, 31].

Regarding the type of mRNA vaccine, participants of studies were vaccinated with the Pfizer-BioNTech BNT162b2 COVID-19 vaccine [17, 34, 32, 29] participants from four studies were immunized with the Moderna mRNA-1273 vaccine [30, 31] while in four studies participants were immunized with either one of the aforementioned vaccines [33, 28, 35, 36]. Most studies assessed the virus-specific T-cell response with the use of QFN-SARS-CoV-2 Starter Pack (Ag1, Ag2) [17, 34, 32, 30, 31, 39, 28, 35, 36], while two studies used the Starter + Extended Pack (Ag1, Ag2 and Ag3) [33, 29]. In the study by Van Praet et al [35] the QFN-SARS-CoV-2 Starter Pack was used and the researchers provided the results from both Ag1 and Ag2 tubes, but separately. However, because at the timepoint we were interested in the QFN T-cell response to both Ags was similar, we decided to include the study and use the results from the Ag2 in the Meta-Analysis [35].

The CASP tools for cohort and case-control studies were used to assess the risk of bias of each of the included studies (Supplementary Tables 2 and 3). All studies fulfilled at least the 60% of criteria of each CASP scale and therefore were considered of “good quality”.

Results of Individual Studies and Syntheses

Τhe results of the included studies are separately reported for each timepoint after vaccination and according to the COVID-19 infection status of the participants (COVID-19-naïve or participants with Hybrid Immunity). Τhe results of the included studies and the results of the synthesis are presented in Figures 2-8.

T-cell response after the Second mRNA vaccine dose for COVID-19-naïve participants: £3 months

The Positivity Rate of QFN-SARS-CoV-2 in in immunocompetent COVID-19 naïve participants, £3 months from the second mRNA vaccine dose was reported in 8 studies [28, 29, 31, 30, 32, 33, 35, 36]. In these studies, the time interval between the completion of the two-dose mRNA vaccination regimen and the T-cell measurement ranged from 2 weeks to approximately three months (13 weeks). The pooled positivity rate (95% CI) at this timepoint from the second mRNA dose was estimated at 91% (88% - 95%). The studies with the greatest % weight was the study by Stieber et al (2023) (45.35%) and by Van Praet et al (2021) (27.05%) [33, 35] (Figure 2a). There was a statistically significant heterogeneity (Q = 42.29; I² = 83.4%; p-value < 0.001), therefore a Random-effects model was taken into consideration Based on the random effects model, the pooled positivity rate (95% CI) at £3 months from the second mRNA vaccine dose was estimated at 81% (71% - 92%) (Figure 2b).

To assess heterogeneity a leave-one-out analysis was performed. The study that influenced heterogeneity the most was the study by Krüttgen et al, (2021 & 2022) [31, 39]. When the analysis was repeated excluding this study, still based on the Random-Effects model, the pooled Positivity Rate (95% CI) £3 months from the second mRNA vaccine dose was 86% (76% - 95%). The heterogeneity was smaller than before but still statistically significant (Q= 28.54; I² = 79.0%; p-value < 0.001). After removing the study by Krüttgen et al (2021 & 2022) [31, 39] from the analysis, the study that influenced heterogeneity the most was the study by Arias-Cabrales et al, 2023 [28]. After removing both studies from the analysis, there was no heterogeneity (Q = 2.02; I² = 0.0%; p-value = 0.731). Based on the meta-analysis of the remaining studies, the pooled positivity rate (95% CI), at £3 months from the second mRNA vaccine dose was estimated at 88% (83% - 93%) (Figure 2c).

For this timepoint after the second dose, out of 188 vaccinees tested with QFN-SARS-CoV-2 Starter Pack (Ag1, Ag2), 149 were positive (79.3%), whereas out of 51 vaccinated individuals tested QFN-SARS-CoV-2 Starter plus Extended Pack (Ag1, Ag2, plus Ag3), 47 were positive (92.2%). The Positivity Rate of the assay differed statistically significantly between studies that used the Starter Pack and studies that used the Extended Pack (Fisher’s exact test, p-value = 0.039). Therefore, a stratified analysis by the type of QFN-SARS-CoV-2 assay (Starter or Starter plus Extended Pack) was conducted. The results of the Stratified analysis per type of assay are presented in Figure 3. The pooled Positivity Rate (95% CI) at £3 months from the second mRNA vaccine 76% (63% - 89%) for QFN-SARS-CoV-2 Starter Pack and 88% (83% - 93%) for QFN-SARS-CoV-2 Starter plus Extended Pack.

The funnel plot for the assessment of possible publication bias for £3months from the second mRNA vaccine dose, showed signs of possible publication bias (Figure 4). To address possible publication bias formally, Egger’s test was performed. The test confirmed that there are signs of publication bias (p-value = 0.017).

T-cell response after the Second mRNA vaccine dose for COVID-19-naïve participants: 3-6 months and >6 months

Two studies reported the Positivity Rate (95% CI) at 3-6 months from the second mRNA vaccine dose in COVID-19 naïve adults [28, 34]. The pooled Positivity Rate (95% CI) of QFN-SARS-CoV-2 at 3-6 months following the administration of the second dose was 59% (45% - 72%). There was no significant heterogeneity in the analysis (Q=1.45; I² =31.2%; p-value=0.228) (Figure 5).

Two studies reported the Positivity Rate (95% CI) at >6 months from the second dose [33, 29]. The pooled Positivity Rate (95% CI) at >6 months from the second mRNA vaccine dose was 79% (66% - 92%). There was no significant heterogeneity in the analysis (Q = 0.00; I² = 0.0%; p-value = 0.965) (Figure 6).

At £3 months after the administration of the second mRNA vaccine dose, a positive response was detected in 196 out of 239 individuals (82.0%), at 3-6 months 29 out of 51 individuals were positive (58.0%), while at >6 months 30 out of 38 individuals had a positive T-cell response (78.9%). The Positivity Rate differed statistically significantly between the three timepoints (Pearson’s chi-square test, p-value < 0.001).

T-cell response after the Third mRNA vaccine dose for COVID-19-naïve participants: ³3 months

Through the databases search, we identified three studies [17, 28, 29] that reported the Positivity Rates of QFN-SARS-CoV-2 after the administration of the third mRNA vaccine dose in COVID-19 naïve participants. Each of these studies belonged to a different Timepoint group [study [28]: 4 weeks after vaccination, study [17]: median (IQR) time from vaccination: 8.08 months (6.97−8.97), study [29]: 3 months post-booster]. Therefore, we conducted a metanalysis in the two of the tree studies categorizing them as ³3 months from vaccination [17, 29]. The pooled Positivity Rate (95% CI) of QFN-SARS-CoV-2 at ³3 months from the receipt of the third mRNA dose was 66% (50% - 82%). There was no significant heterogeneity between the two studies (Q = 2.95; I² = 66.1%; p-value = 0.086) (Figure 7).

T-cell response after the Third mRNA vaccine dose for participants with Hybrid Immunity: ³ 6 months

Among the included studies, we identified five studies that provided results regarding the Positivity Rate of QFN-SARS-CoV-2 for participants with Hybrid Immunity. From these studies, three studies [34, 31, 30] reported the Positivity Rates of the assays after the administration of the second mRNA vaccine dose, for a very limited number of convalescent participants (n£2), so the data for the Positivity Rates of QFN-SARS-CoV-2 were not considered for synthesis of results. The remaining two studies [17, 29] provided results for convalescents for ³6 months after the administration of the third mRNA vaccine dose [study [17]: median (IQR) time from vaccination: 9.55 (7.93−10.14) months, study [29]: 6 months post-booster]. For this specific timepoint, the Positivity Rate (95% CI) of the assay was 81% (67% - 92%). There was significant heterogeneity between these two studies (Q = 4.56; I² = 78.1%; p-value = 0.033) (Figure 8).

Monitoring the virus-specific T-cell responses after vaccination is essential for understanding vaccine-induced immune mechanisms [2]. In addition to this, the duration and the magnitude of these responses are important for determining ongoing vaccination strategies [2]. In the present study, we performed a systematic review and a meta-analysis on the use of QFN-SARS-CoV-2 for the evaluation of cellular immunity after COVID-19 mRNA vaccination in immunocompetent adults, at different time-points after the second or third mRNA vaccine dose. Considering analyzed data from 11 studies, while the QFN assay after 2 or 3 doses of mRNA vaccines detected positive responses in many of the vaccinated immunocompetent individuals at the various time-points post mRNA vaccination, the assay could not detect a T-cell response in a subset of them.

Up to three months after the completion of the primary two-dose mRNA vaccination regimen, in participants without known previous SARS-CoV-2 infection the pooled positivity rate of QFN-SARS-CoV-2, derived from the Random-effects model, was estimated at 81%. Notably, at this timepoint, studies that used the QFN-SARS-CoV-2 Starter plus Extended Pack assay yielded more positive results (92%) than studies that used the QFN-SARS-CoV-2 Starter Pack (79%) in participants without a history or antibody evidence of SARS-CoV-2 infection. QFN-SARS-CoV-2 Starter Pack consists of two Antigen tubes, Ag1 that contains CD4⁺ epitopes derived from the S1 subunit (Receptor Binding Domain) of protein S and Ag2 that contains CD4⁺ and CD8⁺ epitopes derived from the S1 and S2 subunits of protein S [19]. QFN-SARS-CoV-2 Starter plus Extended Pack consists of three Antigen tubes, Ag1, Ag2 plus Ag3 that contains CD4⁺ and CD8⁺ epitopes derived from S1 and S2 subunits of protein S and immunodominant CD8⁺ epitopes derived from the whole viral genome [19]. Therefore, while in our study, we did not assess the Positivity Rate or the IFN-γ levels of each Ag separately, we hypothesize that this difference is due to the inclusion of Ag3. In line with the above observation, other studies that used QFN-SARS-CoV-2 in mRNA vaccinated participants without a previous known SARS-CoV-2 infection, but did not meet the inclusion criteria of this meta-analysis, reported a higher responsiveness in Ag3 as opposed to the other two Ags [40, 19].

While T-cells induced by mRNA COVID-19 vaccination exclusively target the protein S, SARS-CoV-2 natural infection elicits a T-cell response against a wider breadth of antigens (structural and non-structural proteins) derived from the entire viral genome [2, 41]. Therefore, the higher Positivity Rate of QFN-SARS-CoV-2 Extended Pack and possibly Ag3 may be attributed to the misclassification of some participants as COVID-19-naïve as we cannot rule out the possibility that some participants might have had asymptomatic or seronegative SARS-CoV-2 infection [42]. However, higher reactiveness to Ag3 might also be a result of pre-existing cross-reactive immunity against endemic common cold Human Coronaviruses (HCoVs) [43]. Early on in the COVID-19 pandemic, the existence of cross-reactive T-cells was described in unexposed individuals in several studies and it has been estimated that up to 28–50% of individuals may have cross-reactive T-cell memory [44, 43, 45, 46]. It has been reported that these T-cells may be predominately memory T-cells induced by prior HCoVs infection, as HCoVs share to a certain degree some homologous amino acid sequences with SARS-CoV-2 [47, 46]. While the majority of cross-reactive T-cells are CD4⁺ T-cells, less often cross-reactive CD8⁺ T-cell have been also detected [47, 43, 48, 4]. In parallel, regarding hybrid immunity, in a large cohort of mRNA vaccinated Healthcare Workers, higher qualitative and quantitative response to Ag3 was also described in convalescents [43]. Altogether, the above observations suggest that the use of the Starter plus Extended Pack and especially the inclusion of Ag3 might be more useful for detecting the SARS-CoV-2-specific T-cell response post-infection and/or vaccination [40, 49].

Τhe pooled Positivity Rate, at three to six months after the receipt of the second mRNA vaccine dose, declined to 59% and increased to 79% at over six months. A hypothesis for this increase could be either the use of the more sensitive assay with the three Antigen tubes or an asymptomatic infection that may have occurred. In contrast, studies that used the ELISpot and Activated Induced Markers (AIM) T-cell assays have reported that at 6–10 months after a two-dose mRNA vaccination, protein S-reactive T-cells were significantly reduced compared to one month post-vaccination [50, 51].

At the time of the study selection, only two studies that examined T-cell responses with QFN-SARS-CoV-2, in COVID-19 naïve participants, at ≥ three months after the administration of the third mRNA dose, met the inclusion criteria and the pooled Positivity Rate from these studies was estimated at 66%. Due to differences in the time-intervals between vaccination and measurement, and therefore in the categorization of the studies providing data after the second mRNA vaccine dose (≤3, 3–6, >6months) and the third dose (≥3 months), we could not compare directly the findings post the administration of the second and the third mRNA vaccine dose. Likewise, we could not make any direct comparison between the results of participants with hybrid immunity (≥6 months after the third dose) and uninfected participants after the administration of the third dose (≥3 months after the third dose).

Whole-blood based ELISA-IGRAs, like QFN-SARS-CoV-2, are a promising tool for the large-scale study of SARS-CoV-2-specific vaccine-induced T-cell responses [52]. ELISA-IGRAs have the inherent advantages of ease of application, short turnaround times, requirement of minimal equipment and relative low cost, compared to other methods of T-cell assessment [18, 2]. Furthermore, since they require whole blood and not Peripheral Blood Mononuclear Cells (PBMCs), they may reflect better in vivo conditions [2]. In parallel, QFN-SARS-CoV-2 has the additional advantage of a standardized threshold for the detection of a positive T-cell Response [40]. However, due to the novelty of the assay, through the literature research we identified some studies that used their own assay threshold, because the cut-off level might have not been established at the time. To address this issue, we systematically reviewed and analyzed only studies that used a cut-off ≥0.15 IU/mL, as indicated by the manufacturer.

Despite the above advantages, the use of ELISA-IGRAs for studying vaccine-induced immunity has certain limitations. ELISA-IGRAs have a lower sensitivity and specificity compared to other T-cell assays [18]. Some studies have reported a relatively reduced sensitivity, for discriminating vaccinated participants with different infection backgrounds, of the QFN-SARS-CoV-2 assay, compared to the ELISpot and the FC-ICS assays [40, 53]. In addition to this, there are studies using flow-cytometry that report that weeks after receiving the second mRNA dose, a virus-specific CD4⁺ response is induced in approximately 100% of individuals and a virus-specific CD8⁺ response in 70–90% of individuals [47].

Moreover, ELISA-IGRAs do not fully reflect the complicated nature of immunity, as the immune response to virus also includes the B-cell and antibody responses and regarding cellular immunity ELISA-IGRAs do not provide extensive information on the type and quantity of T-cell subsets [2, 18]. Besides, the absence of a detectable T-cell response in the peripheral blood does not necessarily indicate the absence of virus-specific T-cells from lymphoid tissues, in which these T-cells may be rapidly reactivated after exposure to the viral antigens [2].

The interpretation of the present meta-analysis should be done considering the following limitations. Some participants may have had asymptomatic or seronegative SARS-CoV-2 infection and therefore may have been misclassified as COVID-19-naïve in this study. Other limitations include the relatively small number of vaccinated immunocompetent participants that most of the included studies used and the limited number of relevant studies providing data for many time-points post-vaccination. In addition, although we excluded data from immunocompromised patients from the studies, for the rest of the participants we cannot rule out the existence of other comorbidities that may affect the response to vaccination. Finally, there was a limited number of relevant studies regarding the use of the assay in participants with hybrid immunity.

Additionally, significant heterogeneity was detected at ≤3,months post the second dose. However, to address this issue we performed a Random-Effects Model, a leave-out analysis and a stratified analysis by assay type and heterogeneity was reduced. Furthermore, at this time point Egger’s test was applied to examine a possible publication bias and it was found significant, possibly due to restricting the study research process only in electronic databases. Moreover, at 3–6, > 6 months post the second vaccine dose and ≥3 months post the third dose for COVID-19 naïve participants and at ≥6 months after the third dose regarding convalescent participants, only two studies met the inclusion criteria in each timepoint, so publication bias could not be assessed at these timepoints. Finally, in the present study we did not study the vaccine-induced humoral response and its correlation with the T-cell response.

In conclusion, T-cell assays like ELISA-IGRAs could be implemented in the laboratories, along with serological assays, for the assessment of vaccine induced immunity. The QFN-SARS-CoV-2 assay, after the administration of two or three doses of mRNA vaccines, detected positive responses in a certain percentage of the vaccinees. This detection was higher when the QFN-SARS-CoV-2 Starter plus Extended Pack (three Antigen tubes) was used. Possible explanations for the the assay’s negative results in a subset of the participants could be the following: waning immunity, reduced sensitivity of the assay compared to other T-cell assays or lack of induction of T-cell responses to specific antigens in some vaccinees. Further studies examining cell-mediated immunity with longer follow-up periods after vaccination, are required to determine the durability of vaccine-induced T-cell responses.

Author Contributions:

A.M. conceptualized and designed the study. M.M.D and A.M screened publications and extracted data. G.K. performed the statistical analysis. M.M.D. wrote the first draft of the manuscript. A.M. and G.K. critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgments: None

Competing Interest: The authors declare no competing interests.

Funding: No funding was received for conducting this study.

Data availability: The data presented in this study are available on reasonable request from the corresponding author.

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QuantiFERON SARS-CoV-2 assay for the evaluation of cellular immunity after immunization with mRNA SARS-CoV-2 vaccines: A Systematic Review and Meta-Analysis

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