Abstract
Objectives
The SARS-CoV-2 immune response is mediated by both humoral and cellular immunity. In this study, SARS-CoV-2 specific cellular immunity was tested by a novel direct real-time PCR (dRT-PCR) assay, targeting mRNA of CXCL10, and compared with respect to an ELISA measuring interferon gamma (IFN-γ) release.
Methods
Whole blood (Li–He) and serum samples were collected from 92 healthcare workers (HCW), with three doses of homologous (Pfizer/BioNTech, n=74) or heterologous (Pfizer/BioNTech and Vaxzevria or Moderna, n=18) vaccinations. Li–He samples were incubated with SCV2 PANEL-1-T-ACTIVATION (Hyris srl, Lodi, Italy), or CoV-2 IGRA TUBE ELISA (Euroimmune, Lubeck, Germany). CXCL10 mRNA expression was analyzed by bCube/bApp (Hyris), while IFN-γ was evaluated by quant-T-Cell SARS-CoV-2 ELISA (Euroimmune). Anti-SARS-CoV-2 S-RBD IgG levels were measured in sera using a CLIA assay (Snibe, Shenzen, China).
Results
Imprecision of dRT-PCR assay was found to be satisfactory, and the two methods for measuring T cell immunity to SARS-CoV-2 peptides agreed in 82/87 (94.2%) of results. At qualitative dRT-PCR analyses, 81 subjects (93.2%) resulted as reactive to SARS-CoV-2 peptides, 3 (3.4%) were borderline and 3 were negative (3.4%). At univariate and multivariate analyses of quantitative dRT-PCR mRNA of CXCL10 and IFN-γ release results showed no difference between HCW with previous infection, homologous/heterologous vaccination, or demographical features. Anti-SARS-CoV-2 S-RBD IgG was associated with the previous infection and the time between the last vaccination or positivity.
Conclusions
Direct RT-PCR appeared accurate for determining the presence or absence of immunoreactivity of SARS-CoV-2 specific T cells, especially when rapid analyses are required, such as for organ transplantation.
Introduction
During the COVID-19 pandemic, several vaccines based on wild-type (wt) Wuhan-Hu-1, have been successfully developed, reducing SARS-CoV-2 transmission, hospitalizations, severity, and mortality [1], [2], [3], [4]. Among the currently approved COVID-19 vaccines, the two mRNA vaccines BNT162b2 and mRNA-1273 and adenovirus-based vector vaccine ChAdOx1 nCoV-19 ChAdOx1, were the most widely used in Italy [5]. COVID-19 vaccination induces both humoral immunity, mediated by B cell-derived antibodies, and cellular immunity, mediated by T cells [2]. Although neutralizing antibodies represents the first line of anti-viral defence [6], several studies have demonstrated a waning of immune responses and vaccine effectiveness within 6 months after two doses, and again, 6 months after the booster dose [7], [8], [9], [10]. However, not all patients that recover from COVID-19 have detectable neutralizing antibodies [11], which suggests a complex relationship between humoral and cellular response in COVID-19 pathogenesis. Moreover, SARS-CoV-2 variants of concern (VOCs) partially escape humoral but not T-cell responses that are largely conserved against VOCs after prior SARS-CoV-2 infection [12].
Potential therapeutic strategies for COVID-19 include approaches to inhibit, activate or otherwise modulate the immune system. Therefore, it is essential to define the immune response related to specific diseases, especially in well-defined patient cohorts. Some patients may have an impaired T cell response (e.g. transplant patients or immunomodulatory therapy) and therefore require different therapeutic strategies [13, 14].
C-X-C motif chemokine ligand 10 (CXCL10) is a chemokine selectively induced by interferon-γ (IFN-γ) in monocytes, fibroblasts, and endothelial cells upon infection (bacterial and/or viral), in autoimmune pathologies and cancer [15, 16]. Recently, strong evidence showed the upregulation of CXCL10 mRNA in circulating monocytes in response to IFN-γ released by CD8+ T cells and antigen-presenting cells (APC) following SARS-CoV-2 infection and vaccination [17]. In this study, we investigated the association of the immune response after the third dose of vaccine (including both homologous and heterologous vaccination) through a new molecular method in direct real-time PCR amplification (dRT-PCR). We aimed at determining the expression of CXCL10 mRNA, to evaluate the specific response of T cells to SARS-CoV-2 in comparison with the EUROIMMUN CE-IVD device.
Materials and methods
Samples collection
In this study, the population recruited includes healthcare workers (HCW) professionals who joined voluntarily to the study from March 2022 to June 2022. All HCW have been previously vaccinated with three doses of COVID-19 vaccine [Pfizer/BNT162b2 mRNA vaccine, Spikevax (mRNA-1273); ChAdOx1 nCoV-19 vaccine (AZD1222, AstraZeneca)] and underwent a periodical screening for SARS-CoV-2 (every 2 or 3 weeks) by means of rapid antigen testing or molecular rRT-PCR testing. For all patients, demographical characteristics, the date of the vaccination and the type of vaccines were also recorded, as well as the possible date for previous SARS-CoV-2 infection.
Each subject was asked to donate two tubes of whole blood in Li–He (BD Vacutainer LH 102 I.U. 6 mL, Ref. 368886 and a BD Vacutainer LH 68 I.U. 4 mL Ref. 368884) for the assessment of SARS-CoV-2-specific T-cell immunoreactivity, kept at room temperature (RT) until the incubation with peptides; and a serum tube (BD Vacutainer SSTTM II Advance 8.5 mL, Ref. 367953) for the analyses of anti-SARS-CoV-2 S-RBD IgG (S-RBD IgG) antibodies against SARS-CoV-2 (BD Vacutainer SSTTM II Advance 8.5 mL, Ref. 367953).
In this study, we alternatively used the day of the last vaccination or the day of the last positivity (the latter when vaccination occurred after COVID-19 infection) to determine the effect of the time of infection or positivity on cellular or humoral immunity.
Direct real-time PCR amplification of CXCL10
Whole blood samples specific T-cell immunoreactivity against SARS-CoV-2 peptides was analysed by the Hyris system [composed of SCV2 PANEL 1 T ACTIVATION kit, bKIT™ Immunofinder dqTACT, the bCUBE system and 32-well and 16-well cartidge and bAPP v1.5.12 (https://bapp.hyris.net/)]. Through the SCV2 PANEL 1 T ACTIVATION kit (Ref. HK020X050), the heparinized blood is stimulated within 6 h after collection, with a pool of peptides overlaying the protein sequence spike (S) (pool ONE). T-cell activation is evaluated following an overnight incubation of 16–18 h. In particular, the kit stimulates the release of IFN-γ of antigen-specific T cells, which in turn attract monocytes, that release a large amount of CXCL10. Since CXCL10 mRNA expression is directly related to the activation of the antigen-specific T lymphocytes, this can be used to quantify immunity cell-mediated by dRT-PCR. The use of a second kit, bKIT™ Immunofinder dqTACT (Ref. HK032X300) is required to detect the mRNA of CXCL10. The direct RT-PCR test which is used in this type of analysis was developed and patented by Hyris (Hyris srl, Lodi, Italy). Direct RT-PCR reaction allows to reverse transcribe and amplify CXCL10 mRNA in a single reaction. The qualitative results are directly reported in the bApp through an artificial intelligence algorithm based on the Ct thresholds. In qualitative analysis, the samples can be defined as “responsive”, “bordeline” or “not responsive” compared to samples stimulated with non-SARS-CoV-2 peptides (pool NEG). This system has been recently validated elsewhere [18].
In addition to the amplification of CXCL10 mRNA by pool ONE, we were able to test the efficiency of other 3 pools of peptides combinations (used as stimulus), not validated for the CE-IVD market yet. Pool B included the nucleocapside (N) SARS-CoV-2 peptides, pool C included peptides from the regions of the protein S mutated in omicron variant (BA.1) of SARS-CoV-2 virus (pool C), and pool D included other peptides of the protein sequence of S protein (Figure 1). Notably, the Hyris system was not clinically validated for pool B, pool C and pool D, which a technology under development.
Schematic diagrams of the SARS-CoV-2 virus genome and designed pool peptides. (A) SARS-CoV-2 virus genome. S encodes the N-terminal domain (NT), receptor-binding domain (RBD), subdomains 1 and 2 (SD1 and SD2), fusion loop (FP), heptad repeat 1 and 2 (HR1 and HR2), transmembrane domain (TM). (B) Designed pool peptides from the top-down: pool ONE (whole S wt protein peptides), pool B (whole N protein peptides), pool C (fragment of S Omicron variant, BA.1), pool D (fragment matching pool C, of S wt protein regions).
Assessment of analytical precision
Nine blood samples collected in Li–He tubes taken from 3 donors were used to evaluate the overall analytical precision of Hyris system for detecting CXCL10 mRNA. For each subject, the 3 samples were independently processed and stimulated overnight using pool ONE as above specified and then analyzed by dRT-PCR for detecting CXCL10 mRNA expression.
Specific T-cell immuno-reactivity analyses for SARS-CoV-2 peptides using ELISA assay
Li–He blood samples were distributed and incubated for 20–24 h (+37 °C ± 1 °C) in 3 different stimulation tubes: (1) CoV-2 Interferon Gamma Release Assay (IGRA) BLANK, (2) CoV-2 IGRA TUBE, (3) CoV-2 IGRA STIM. Subsequently, the interferon-gamma concentration of the plasma samples is determined using the EUROIMMUN Quan-T-Cell ELISA kit (EUROIMMUN code no. EQ 6841-9601). The multistep interpretation of the results enclosed initially the verification of sufficient immune stimulation (STIM control value subtracted of the BLANK concentration). Subsequently, the IFN-γ concentration (CoV-2 IGRA TUBE) was evaluated to obtain information on the immune reaction to the SARS-CoV-2 virus or to the vaccination. The manufacturer’s limit ranges for results interpretation are: negative: <100 mIU/mL; borderline: 100–200 mIU/mL; positive: >200 mIU/mL (Insert ET_2606_A_UK_C01, version 2021-08-26).
Determination of anti-SARS-CoV-2 S-RBD IgG by CLIA assay
The antibody anti-SARS-CoV-2 IgG against the RBD portion of the Spike (S) protein was determined by Maglumi 2000 plus (Snibe Diagnostics, Shenzhen, China), validated elsewhere [19].
Statistical analyses
GraphPad Prism version 9.4 for Windows (GraphPad Software, LLC) was used for graphical plots of results. Stata 16.1 (Statacorp, Lakeway Drive, TX, USA) was employed for descriptive statistics, univariate analyses (Kruskall-wallis, Student᾽s t-test and Fishers’ exact test) and for multivariate analyses (linear regression), performed using log transformed anti-SARS-CoV-2 S-RBD IgG Ab and IFN-γ levels, and −
Ethical approval
The study was conducted in accordance with the Declaration of Helsinki, and the Institutional Review Board of the University of Padova (protocol no. 27444).
Results
Study population
Table 1 reports the characteristics of the 92 individuals included in this study, subdivided by males and females. Among the 92 HCW included in the study, 5 donated two samples, the second one 1 month apart. Thus, in total, 97 samples were available for testing T cell response to SARS-CoV-2 peptides. The time in days between the last vaccination or positivity and the evaluation of T cells response to SARS-CoV-2 peptides differs between individuals with homologous vaccination (157.5
Characteristics of the individuals included in the cohort study. Table represents the percentage of all HCW, female and male. The age is expressed in years, the time in days.
| Characteristics | All subjects | Female | Male | p-Valuea |
|---|---|---|---|---|
| n (%) | 92 (100%) | 69 (75.0%) | 23 (25.0%) | |
| Age, years (mean ± DS) | 46.6 ± 13.1 | 46.8 ± 12.3 | 45.7 ± 15.4 | χ2=0.159, p=0.690b |
| Presence of SARS-CoV-2 previous infection | 27/92 (29.3%) | 21/69 (30.4%) | 6/23 (26.1%) | p=0.795c |
| Individuals with at least three vaccinations doses | 90/92 (97.8%) | 68/69 (98.6%) | 22/23 (95.6%) | p=0.999c |
| Homologous vaccination | 74/92 (80.4%) | 58/69 (84.1%) | 16/23 (69.5%) | p=0.141c |
| Heterologous vaccination | 18/92 (19.5%) | 11/69 (15.9%) | 7/23 (30.4%) | |
| Time between the last vaccination or positivity (mean ± DS) | 145.2 ± 55.2 | 140.4 ± 54.2 | 159.6 ± 56.9 | χ2=1.251, p=0.263b |
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aCalculated between female and male subjects, bKruskall-Wallis equality-of-population rank test, cFisher’s exact test.
Direct real-time PCR amplification of CXCL10 – qualitative results
The overall qualitative results for pool ONE, obtained with the Hyris system for the 97 samples were: 3 negative, 3 borderline, and 89 positive; one sample, resulting inconclusive, was not re-analyzed since after stimulation with SARS-CoV-2 peptides the sample was not immediately frozen. Interestingly, the negative HCW underwent vaccination 113, 152 and 184 days before sample collection and all were not previously affected by COVID-19.
The ELISA IFN-γ measurements were performed in 93 of the 97 samples (95.9%). Five out of 93 samples (5.4%) resulted inconclusive. Considering the manufacturer’s declared thresholds, in 1 sample measured values were between 100 and 200 IU/L (borderline result), while in 87 samples IFN-γ levels were above 200 IU/L and were then considered positive. Similarly, the individual with borderline results underwent vaccination 222 days before samples collection.
Hence, the following analyses of methods agreement, reported in Table 2, were performed considering a total of 87 results. The two methods for measuring T cell immunity to SARS-CoV-2 peptides agreed in 82/87 (94.2%) of results.
Comparison of qualitative results between the studied assays. The two methods for measuring T cell immunity to SARS-CoV-2 peptides, Hyris T-cell and ELISA IFN-γ, agreed in 94.2% of results.
| Results | CXCL10 mRNA (pool ONE) | Elisa IFN-γ | Anti SARS-CoV-2 S-RBD IgG |
|---|---|---|---|
| Positive | 81 (92.1%) | 86 (98.9%) | 87 (100%) |
| Borderline | 3 (3.4%) | 1 (1.1%) | 0 (100%) |
| Negative | 3 (3.4%) | 0 (0%) | 0 (100%) |
| Total | 87 (100%) | 87 (100%) | 87 (100%) |
Direct real-time PCR amplification of CXCL10 – quantitative analyses
The bApp offers the possibility to obtain the raw cycle threshold (Ct) values of both the target and the housekeeping genes (CXCL10 and Actin, respectively) for all the studied pools of peptides (pool ONE, pool B, pool C and pool D). In addition, results were available for both the stimulated and unstimulated mixtures. Then, it was possible to estimate the relative quantification of the PCR signal of the target transcript by the method of the 2−∆∆Ct as specified by Livak et al. [20] and that consider both the stimulated (pool ONE or pool B, pool C and pool D) and unstimulated mixtures (pool NEG). However, one technical limit was observed with this calculation method: in some samples, the unstimulated mixture (pool NEG) resulted in a CXCL10 expression equal to or above the Ct limit (which was 45, indicating that the gene expression was below the limit of detection). This limit made 2−∆∆Ct the results of the estimation unrealistic. Thus, a correction factor was applied to samples in which pool NEG CXCL10 was not detected. To estimate this correction factor we first calculated the
Precision study results
The precision of the three analysed samples was evaluated with respect to the 2−ΔΔCt method. The mean values of the triplicates were 32.8, 25.1 and 67.7, measured with a CV of 10.7, 18.5 and 28.0%, respectively.
Clinical validation results
Figures 2 and 3 report the results for 2−∆∆Ct of CXCL10 mRNA after stimulation with pool ONE, IGRA ELISA IFN-γ measurements, and Anti-SARS-CoV-2 S-RBD IgG, subdivided by gender, individuals with and without previous COVID-19 infection, and homologous/heterologous vaccinations and the scatter diagrams with age. Table 3 reports the univariate and multivariate analyses of log-transformed of 2−∆∆Ct CXCL10 mRNA after pool ONE stimulation (log2), IGRA ELISA IFN-γ measurements (log10), and Anti-SARS-CoV-2 S-RBD IgG levels (log10) and the studied variables. Despite the 2−∆∆Ct CXCL10 mRNA was not correlated with age, when gender was considered the correlation between 2−∆∆Ct CXCL10 mRNA and age was significant for females (Spearman’s r = −0.462, p = 0.023), but not for males (Spearman’s r = −0.144, p = 0.222). The correlation between 2−∆∆Ct CXCL10 mRNA and IFN-γ levels was significant (Spearman’s r = 0.302, p = 0.003), while the correlation between 2−∆∆Ct CXCL10 mRNA and anti-SARS-CoV-2 S-RBD IgG was not significant (Speaman’s r = 0.101, p = 0.324). Differently, the levels of IFN-γ levels and anti-SARS-CoV-2 S-RBD IgG were significantly correlated (Spearman’s r = 0.407, p < 0.001).
Dotplots of dRT-PCR CXCL10 mRNA (pool ONE) (A), IGRA IFN-γ (B) and anti-SARS-CoV-2 S-RBD IgG (C) results, subdivided by males and females. Scatterplots of HCW age and dRT-PCR CXCL10 mRNA (pool ONE) (D), IGRA IFN-γ (E) and anti-SARS-CoV-2 S-RBD IgG (F) results, with different symbols used for males and females, as reported in pictures. Dotted lines represent assays thresholds.
Dotplots dRT-PCR CXCL10 mRNA (pool ONE) (A and B), IGRA IFN-γ (B and E) and anti-SARS-CoV-2 S-RBD IgG (C and F) results, reported subdivided by homologous/heterologous vaccination, and the presence or absence of previous COVID-19. For each graph, y-axes are in log scales. Dotted lines represent assays thresholds.
Univariate (linear regression) and multivariate analyses (multiple regression) analyses.
| Univariate analyses variables | dRT-PCR pool ONE −ΔΔCTa | IGRA (log10 of IFN-γ) | log10 Anti-SARS-CoV-2 S-RBD IgG |
|---|---|---|---|
| Age | β=−0.034, p=0.069 | β=−0.003, p=0.455 | β=−0.001, p=0.929 |
| Sex | β=−0.242, p=0.667 | β=−0.146, p=0.212 | β=−0.028, p=0.822 |
| Presence of SARS-CoV-2 previous infection | β=0.994, p=0.056 | β=0.169, p=0.123 | β=0.540, p<0.001 |
| Heterologous vaccination | β=−0.268, p=0.667 | β=0.087, p=0.507 | β=0.182, p=0.196 |
| Time between the last vaccination or positivity | β= 0.001, p=0.940 | β=−0.001, p=0.165 | β=−0.003, p<0.001 |
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| Multivariate analyses variables | |||
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| Age | β=−0.029, p=0.128 | β=−0.002, p=0.525 | β=0.002, p=0.654 |
| Sex | β=−0.234, p=0.690 | β=−0.141, p=0.256 | β=0.059, p=0.609 |
| Presence of SARS-CoV-2 previous infection | β=0.911, p=0.103 | β=0.118, p=0.320 | β=0.464, p<0.001 |
| Heterologous vaccination | β=−0.131, p=0.855 | β=0.063, p=0.680 | β=−0.002, p=0.988 |
| Time between the last vaccination or positivity | β=0.002, p=0.690 | β=−0.001, p=0.545 | β=−0.002, p=0.017 |
- a
Equal to log2 of 2−ΔΔCT. p-Values below 0.05 are marked as bold.
Supplementary Table 1 summarizes the results for 2−∆∆Ct pool ONE, 2−∆∆Ct pool B, 2−∆∆Ct pool C, 2−∆∆Ct pool D, IFN-γ and anti-SARS-CoV-2 S-RBD IgG levels.
Discussion
The rapid spread of SARS-CoV-2 infection worldwide has forced the development of vaccines to reduce viral transmission and to prevent the risk of severe illness [21]. In 2021 several European governments recommended discontinuing using of the ChAdOx1 nCoV-19 vaccine (AZD1222, AstraZeneca) in the young and middle-aged population. For this and other reasons, it has been an enhancement of heterologous vaccination schedules despite the lack of information regarding immunogenicity and safety features [22]. Nowadays, several studies have provided reassurance that there are multiple appropriate options to complete COVID-19 immunization in individuals, showing evidence of mixed adenoviral and mRNA schedules as being safe, tolerable, and immunogenic alternatives to homologous programs [23]. The protection against infection depends on the complex relationship between humoral and cellular response in COVID-19 and for evaluating the protection against infection, it is of relevance the assessment of humoral immunity and of cellular response to SARS-CoV-2 [9]. It has been demonstrated that in vaccinated subjects without previous SARS-CoV-2 infection, the Ab titres start to dampen after 6 months post-vaccination, in subjects with previous infection antibody protection is much more extensive and lasting [24], [25], [26]. Vaccine immunization was also demonstrated to stimulate immunological T cell memory, which was able to recognize variants from Alpha to omicron [12]. Long-lasting memory T cell-mediated protection after infection or vaccination is rather complicated as it may involve SARS-CoV-2-specific cytotoxic T lymphocytes or cross-reactivity with previously infected seasonal coronaviruses [27]. Besides, immunocompromised patients have reduced immunity, associated with a faster decline in the antibodies levels [28]. Therefore, several studies have evaluated the level of immune response in well-defined patient cohorts individuals, treated or not treated with drugs therapies, including subjects with homologous and heterologous vaccination [29].
Considering a cohort of 92 HCW, this study provides insights into the immunogenic outcome of three standard doses of homologous (n = 74) (BNT162b2) or heterologous (n = 18) vaccines (BNT162b2, ChAdOx1 nCoV-19 and Spikevax). In this cohort, a previous diagnosis of SARS-CoV-2 infection by routine NAAT screening was officially recorded in 29.3% of individuals. The cellular immune response was investigated with two types of assays, presenting different technologies. Indeed, a new molecular SARS-CoV-2 dRT-PCR assay (Hyris system), based on CXCL10 mRNA amplification, was evaluated with respect to a SARS-CoV-2 Interferon Gamma Release Assay (IGRA), based on ELISA. Noteworthy, in qualitative analyses, the two methods agreed in 82/87 (94.2%) of the data demonstrating high comparability. This percentage is in agreement with (but higher than) the concordance rate (71.2%) previously reported by the findings of Aiello et al. [30], who highlighted that a discrepancy in cellular immunity could be due to the composition of the different peptides and to assays technology. A similar reason could account for the non-complete agreement found in our data between dRT-PCR and the IGRA assays.
The dRT-PCR system allowed obtaining also quantitative results by means of 2−∆∆Ct. For mRNA of CXCL10 after stimulus with pool ONE, the performances for repeatability (measured including also the sample preparation process) were all below 28%, in line with results already obtained from other Nucleic Acid Amplification Test (NAAT) from Rao et al., in which the PCR efficiency issues limited results reproducibility [31].
In addition to the CE-IVD market-approved stimulus (pool ONE), we were able to study additional pools of peptides (pool B, C and D), experimentally developed by manufacturers, all currently still marked as research use only. For these peptide pools, we produced only descriptive results (Supplementary Table 1), without proving the effective performances, since this was beyond the aims of this study.
The association of gender, age, presence of SARS-CoV-2 previous infection, and the time between the last vaccination or positivity with the cellular and humoral immune response to SARS-CoV-2 was studied for CXCL10 mRNA expression (dRT-PCR), IFN-γ IGRA (ELISA) and anti-SARS-CoV-2 S-RBD IgG (Figures 2 and 3), by univariate and multivariate analyses. Both analyses highlighted that significant associations exist for anti-SARS-CoV-2 S-RBD IgG and the presence of SARS-CoV-2 previous infection and the time between the last vaccination or positivity; none of these variables were associated with the cellular immune response. These findings were in agreement with results already reported in the literature by Vogel et al., where long-term maintenance of SARS-CoV-2 spike-reactive T cells after homologous and heterologous vaccination was demonstrated studying IFN-γ response [32]. Therefore, we evaluate immunity in heterologous/homologous vaccination. According to Vogel et al., results for humoral and cellular immunity demonstrated a noninferiority of heterologous vs homologous vaccination [32]. However, different to findings reported in the literature by other Authors [22, 33, 34], in our results, neither cellular nor humoral immunity levels were associated with the homologous/heterologous vaccination. This discrepancy could be explained by the limited number of subjects with heterologous vaccination and the narrow age range of the cohort.
The results obtained with pool B, C and D are all summarized in Supplementary Table 1. Of interest, Spearman’s correlations between CXCL10 mRNA expression (pool ONE) and other pools were: r=0.165 (p=0.137) for pool B, r=0.558, p<0.001 for pool C, r=0.475, p<0.001 for pool D. Spearman’s correlation between other pools were: pool B and C, r = 0.339, p = 0.0018; pool B and D, r=0.153, p<0.165, pool C and D, r=0.521, p<0.001. These results are expected, since pool A and D, being both designed starting by the SARS-CoV-2 Wuhan strain (Figure 1). Similarly, pool C, which was designed starting from the Omicron variant BA.1 (Figure 1), highly correlated with pool A and D. Differently, pool B, designed starting from the N peptides of the SARS-CoV-2 Wuhan strain (Figure 1), appeared to be quite inefficient in detecting the presence of a previous SARS-CoV-2 infection, being not different between individuals with or without previous COVID-19 (
This study presents some limitations, such as the low number of HCWs included, the low proportion of individuals with heterologous/homologous vaccination, and the ratio of females/males individuals, which is quite unbalanced. Further, we did not evaluate the impact of total white blood cells on the assay performances and the measured cellular response. On the other side, the strengths of this study is the evaluation of cellular immune response by two different assays based on different technologies.
Conclusions
The findings made in the present study generate further proof of the ability of vaccines to stimulate B and T cells response, which, in addition to natural immunity, synergically contributes to protect against the SARS-CoV-2 infection. These results for T-cell response were confirmed by two different analytical assays. We also demonstrated the feasibility of routinely testing cellular immunity by using a new molecular test based on dRT-PCR. This latter test system is rapid, with a minimal restrained instrument size, and includes the possibility of evaluating results remotely, using an artificial intelligence-based system for offering real-time results, without the requirements of operator intervention of interpretation. These features would enable the assessment of COVID-19 immunological responses in some clinical settings, such as patients receiving transplantation or during immuno-therapies, where rapid tests could be pivotal for real-time patient evaluation.
Acknowledgments
The Authors thank Hyris Ltd, Euroimmun and Snibe Diagnostics for kindly supplying reagents without in any way influencing the study design and data analysis.
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Research funding: None declared.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: The authors declare no conflict of interest
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Informed consent: Not applicable.
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Ethical approval: The study was conducted in accordance with the Declaration of Helsinki, and the Institutional Review Board of the University of Padova (protocol no. 27444).
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