key: cord-1038109-wrh0bx93
authors: Azgomi, Mojtaba Shekarkar; La Manna, Marco Pio; Badami, Giusto Davide; Ragonese, Paolo; Trizzino, Antonino; Dieli, Francesco; Caccamo, Nadia
title: A Rapid and Simple Multiparameter Assay to Quantify Spike-Specific CD4 and CD8 T Cells after SARS-CoV-2 Vaccination: A Preliminary Report
date: 2021-10-29
journal: Biomedicines
DOI: 10.3390/biomedicines9111576
sha: 5af60375fc4572e9ee73896e7c5f0a57a28fc11a
doc_id: 1038109
cord_uid: wrh0bx93

mRNA and Adenovirus vaccines for COVID-19 are used to induce humoral and cell-mediated immunity, with the aim to generate both SARS-CoV-2 B and T memory cells. In present study, we described a simple assay to detect and quantify Spike-specific CD4(+) and CD8(+) T cell responses induced by vaccination in healthy donors and in subjects with B cell compart impairment, in which antibody response is absent due to primary immunodeficiencies or CD20 depleting therapy. We detect and quantified memory T cell immune responses against SARS-CoV-2 evocated by vaccination in both groups, irrespective to the humoral response. Furthermore, we identified TNF-α as the main cytokine produced by T memory cells, after antigen-specific stimulation in vitro, that could be considered, other than IFN-γ, an additional biomarker of induction of T memory cells upon vaccination. Further studies on the vaccine-induced T cell responses could be crucial, not only in healthy people but also in immunocompromised subjects, where antigen specific T cells responses play a protective role against SARS-CoV-2.

After more than 2 years, a syndrome case by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 4 million deaths worldwide, and it still is a prevalent outbreak [1] . To date, different licensed vaccines for COVID-19 with different techniques are available, and all presumably induce both humoral and cell-mediated immunity, both of which are required for recovery following SARS-CoV-2 infection. In addition, patients with more severe diseases develop neutralizing antibodies (NAb) that correlate with viral load [2, 3] . Still, on the other hand, SARS-CoV-2 can spread from cell to cell without exposure to the extracellular environment [4] , limiting the role of NAb in reducing viral spread within the host. For this reason, T cells could be important mediators of the protective host response to SARS-CoV-2 infection. Hence, there is an urgent need to consider the role of SARS-CoV-2-specific T cell immune response after vaccination, analyzing either CD4 + T cells, which help B cells for antibody production, or CD8 + T cells that kill virus-infected cells [5, 6] . Therefore, a simple and rapid assessment of T cell immune response after vaccination remains a challenge. Using flow cytometry, Riou and colleagues described a rapid assay to qualitatively and quantitatively measure SARS-CoV-2-specific CD4 + T cell responses in 31 healthcare workers [7] . Despite several studies demonstrating the feasibility of detecting SARS-CoV-2-specific CD4 + and CD8 + T cells, most of them have been performed in SARS-CoV-2-infected individuals and using different techniques. Still, there is very limited knowledge on similar assays performed after vaccination. Here, we report a rapid (~18 h) method to monitor SARS-CoV-2-specific T cell responses after vaccination independently from NAb blood concentration, which is alsousable in patients with primary or secondary B cell defects.

This assay relies on simultaneous expression of three cytokines (IL-2, IFN-γ and TNF-α) after 18 h of stimulation in vitro with a pool of lyophilized peptides, consisting mainly of 15-mer sequences with an 11 amino acid overlap, covering the immunodominant sequence domains of the spike glycoprotein of SARS-CoV-2 (PepTivator ® SARS-CoV-2 Prot_S, Miltenyi Biotec, Surrey, UK). The study was approved by the Ethical Committee of the University Hospital. Peripheral blood mononuclear cells (PBMCs) were obtained from n = 41 fully vaccinated (mRNA vaccine BNT162b2, Pfizer/BioNTech, Mangonza, Germany) individuals, 15 to 21 days after receiving the second dose vaccine. Asymptomatic subjects negative for SARS-CoV-2 with a real-time PCR test were enrolled in the study.

The enrolled subjects (n = 40), were divided into four groups; the first three groups were composed of healthy donors (n = 28) with different titers of SARS-CoV-2 NAbs (SARS-CoV-2 Trimeric S IgG) at the time of venipuncture: G1 with a level of NAb < 390 BAU/mL, G2 with 390 < NAb < 1040 BAU/mL, G3 with NAb > 1040 BAU/mL. The fourth group (G4) was composed of patients (n = 12) without functional B cells and no NAb in peripheral blood (six patients with multiple sclerosis in therapy with ocrelizumab and six patients with primitive B cell deficiencies). The demographic characteristics of the groups are reported in Table 1 while the specific clinical and therapeutic characteristics of G4 patients are reported in Table 2 . PBMCs were stimulated for 18 h at 37 • C 5% CO 2 in RPMI1640 complete medium, witha spike-specific peptide pool (1 µg/mL) at 1 × 10 6 cells/mL. RPMI or ionomycin/PMA were included in each sample as negative or positive controls, respectively. Brefeldin-A (10 µg/mL) was added after 2 h.

After 18 h of stimulation, cells were harvested and stained, first with live/dead marker (Zombie dye, Biolegend San Diego, CA, USA) then with mAb anti-human CD3PerCP-Vio ® 700, mAb anti-human CD4 PE-Vio ® 770 and mAb anti-human CD8 APC. After surface staining, cells were fixed, permeabilized and stained at room temperature for 30 min with mAb to anti-human IL-2 APC-Vio ® 770, mAb anti-human IFN-γ FITC and mAb anti-human TNF-α PE. Samples were acquired on a FACSARIA II flow cytometer (BD Bioscience San Jose CA, USA) and analyzed using FlowJo v10 (BD Bioscience San Jose CA, USA).

The gating strategy is shown in Figure 1A . The threshold for positivity for spikespecific CD4 + T cell responses (>0.02%) and antigen-specific CD8 + T cell responses (>0.05%) was set according to Dan et al. [7] [8] [9] and calculated using the median two-fold standard deviation of all negative controls measured. GraphPad software was used to perform statistical analysis, and the groups were analyzed by using a Kruskal-Wallis test with Dunn's correction and a Pearson correlation test. Quantification of spike-specific CD4 + and CD8 + T cells after SARS-CoV-2 vaccination. Gating strategy used to identify spike-specific CD4 + and CD8 + T cells and to detect their cytokine expression in response to spike-derived peptides. (A)Cumulative frequency and distribution of spike-specific CD4 + and CD8 + T cells (B) in SARS-CoV-2-vaccinated individuals (n = 40). Cut-off for positivity was set at <0.02 for CD4 + T cells and <0.05 for CD8 + T cells. Analysis of distinct cytokine expression by spike-specific CD4 + (C) and CD8 + (D) T cells using Kruskal-Wallis test with Dunn's correction. ** p < 0.01; *** p < 0.001; ns: not significant. 

The proportion of vaccinated subjects positive for spike-specific memory CD4 + and CD8 + T cells, measured as the frequency of CD4 + and CD8 + T cells simultaneously expressing IL-2, IFN-γ and TNF-α, was 90% (36/40) and 70% (28/40), respectively ( Figure 1B) . Indeed, both the spike-specific CD4 + and CD8 + responses were characterized by very faint (if any) IL-2 expression and very low IFN-γ expression, but were enriched in cells expressing TNF-α ( Figure 1C,D) . Thus, and also in agreement with the study of Riou and colleagues [7] , in our assay, TNF-α was the predominant cytokine produced, either by spike-specific CD4 + or CD8 + T cells, suggesting that TNF-α could be a more reliable biomarker than any other cytokine to detect spike-specific T cells in response to vaccination.

To study the correlation between the spike-specific T cell response and the NAb response, we stratified vaccinated healthy donors into three groups according to the titer of SARS-CoV-2NAbs: G1, <390 BAU/mL; G2, 390-1040 BAU/mL; G3, >1040 BAU/mL. As shown in Figure 2A ,B, the spike-specific CD4 + and CD8 + responses were of similar magnitude irrespective of the NAb titers in the G1, G2 and G3 groups. Accordingly, we did not find significant correlation between frequencies of spike-specific CD4 + and CD8 + T cells with NAb titers ( Figure 2C,D) , at least limited to those vaccinated subjects for whom precise antibody titers were available.

Finally, we analyzed the spike-specific T cell response in a group of subjects affected by primary or secondary B cell deficiencies, that had been vaccinated with the Pfizer/BioNTech BNT162b2 mRNA vaccine. Despite failing to produce spike-specific antibodies (Table 3) , the B cell-deficient subjects showed a measurable spike-specific T cell response, comparable with that of the other groups.

of SARS-CoV-2NAbs: G1, <390 BAU/mL; G2, 390-1040 BAU/mL; G3, >1040 BAU/mL. As shown in Figure 2A ,B, the spike-specific CD4 + and CD8 + responses were of similar magnitude irrespective of the NAb titers in the G1, G2 and G3 groups. Accordingly, we did not find significant correlation between frequencies of spike-specific CD4 + and CD8 + T cells with NAb titers (Figure 2C,D) , at least limited to those vaccinated subjects for whom precise antibody titers were available. Figure 2 . Correlation of spike-specific CD4 + and CD8 + T cells with spike-specific NAb titers. Frequencies of spike-specific CD4 + (A) and CD8 + (B) T cells upon stratification of vaccinated individuals in 3 different groups according to their SARS-CoV-2 NAb titers (groups G1 to G3). Pearson correlation coefficient with one-tailed P-value and 90% confidence interval between titers of SARS-CoV-2 NAbs and the frequency of spike-specific CD4 + (C) and CD8 + (D)T cells. Figure 2 . Correlation of spike-specific CD4 + and CD8 + T cells with spike-specific NAb titers. Frequencies of spike-specific CD4 + (A) and CD8 + (B) T cells upon stratification of vaccinated individuals in 3 different groups according to their SARS-CoV-2 NAb titers (groups G1 to G3). Pearson correlation coefficient with one-tailed P-value and 90% confidence interval between titers of SARS-CoV-2 NAbs and the frequency of spike-specific CD4 + (C) and CD8 + (D)T cells. 

Much scientific evidence suggests that the cell-mediated response assumes relevant importance for an effective immune response against SARS-CoV-2 virus [10] . One study, in particular, showed that in cancer patients, the establishment of the cell-mediated response induced by the vaccine, even in the absence of a corresponding antibody response, was sufficient to confer protection against infection [11] . Another study has highlighted the establishment of the cellular immune response against SARS-CoV-2 in subjects vaccinated with BNT162b2Pfizer/BioNTech in the absence of an antibody response at the time of the assessment of adaptive immunity, after the second injection [12] . Furthermore, many studies have shown that mRNA vaccines induced a reduced humoral response in patients with acquired immunodeficiencies due to hematological diseases [13] or immunosuppressive therapies, including the use of biological drugs such as rituximab [14, 15] . Finally, the Pfizer vaccine also provokeda cell-mediated immune response in subjects with primary immunodeficiencies affecting the B compartment [16] . This literature shows the potential usefulness of a test to evaluate the efficacy of vaccination by measuring the cellular immune response.

Some attempts have been made in this direction, also considering evaluating IFNγ using IGRA tests [17] . However, the T cells' response is expressed through different activation pathways, and the evaluation of a single cytokine may not fully reflect the state of activation of this compartment. A multiparametric flow cytometry test, such as the one proposed in this communication, through the evaluation of the simultaneous expression of TNF-α, IL2 and IFN-γ can identify more completely the presence of a cell-mediated immune response induced by vaccination regardless of the evaluation of the humoral response.

Our study shows that vaccination with Pfizer gives a robust cell-mediated immunological memory against spike protein antigens, independently from the titer of NAb, meaning that the T cell-mediated specific immune response against SARS-CoV-2 caused by vaccination can develop independently from B cell response. This finding highlights the importance of the cell-mediated immunity against SARS-CoV-2 induced by vaccination and identifies TNF-α as the main product of T cells of vaccinated subjects after specific stimulation in vitro, making this cytokine a correlate of successful vaccination. In conclusion, we have reported a proof-of-concept study describing a simple and easy assay to detect and quantify spike-specific CD4 + and CD8 + T cell responses induced by vaccination. Although our study has many limitations (small number of tested individuals, optimization and validation), it provides a tool to monitor the immunogenicity of SARS-CoV-2 vaccines and study the correlation between the quantity and quality of B and T cell-mediated responses and protection. Finally, studying vaccine-induced T cell responses may be of value in those subjects with B cell depletion following ocrelizumab or due to primary immunodeficiencies, in which serological responses are impaired, but T cell responses are preserved.

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Data Availability Statement: The Data will be available upon reasonable request to the corresponding author.

The authors declare no conflict of interest.