Long-Lasting T Cell Responses in BNT162b2 COVID-19 mRNA Vaccinees and COVID-19 Convalescent Patients

Abstract

The emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made it more difficult to prevent the virus from spreading despite available vaccines. Reports of breakthrough infections and decreased capacity of antibodies to neutralize variants raise the question whether current vaccines can still protect against COVID-19 disease. We studied the dynamics and persistence of T cell responses using activation induced marker (AIM) assay and Th1 type cytokine production in peripheral blood mononuclear cells obtained from BNT162b2 COVID-19 mRNA vaccinated health care workers and COVID-19 patients. We demonstrate that equally high T cell responses following vaccination and infection persist at least for 6 months against Alpha, Beta, Gamma, and Delta variants despite the decline in antibody levels.

Keywords: BNT162b2; Covid-19; T cell mediated immunity; humoral immunity; vaccine.

Figure 1

Optimization of AIM test and the kinetics of cytokine mRNA expression of stimulated PBMCs. PBMCs collected from five vaccinees were stimulated with 10 µg/ml pf tetanus toxoid (TET), 1 µg/ml PHA, and 0.5 µg/ml SARS-CoV-2 Wuhan Hu virus spike peptide pool (S_wt) for 4, 8, 16, 24, and 48h. (A) After 24h and 48h stimulation, specific T cell responses were measured with flow cytometry using expression of CD69 and CD134 as a marker for CD4⁺ T cell activation, and CD69 and CD137 as a marker for CD8⁺ T cell activation. Stimulation index (SI) was calculated by dividing the percentage of AIM⁺ cells after TET, PHA, or S_wt stimulation with the percentage of AIM⁺ cells after DMSO stimulation. (B) Cytokine mRNA expression was analyzed with RT-qPCR from total cellular RNA isolated from cells collected at different times after stimulation. RNA levels at different time points were compared to corresponding DMSO treated samples. The geometric means and standard deviations are show in the figures.

Figure 2

SARS-CoV-2 spike-specific CD4+ and CD8+ T cell responses after BNT162b2 vaccination. Peripheral blood mononuclear cells (PBMCs) collected from health care workers (HCWs) at different times after two doses of BNT162b2 vaccine were stimulated with wild type (wt) SARS-CoV-2 spike protein-specific peptides for 48 h. (A) Representative graphs from one vaccinated individual illustrating the gating strategy used for all participants in flow cytometric analysis in T cell activation induced marker (AIM) assay. Antigen-specific CD4⁺ responses were defined with the expression of CD69⁺ and CD134⁺, and CD8⁺ responses with the expression of CD69⁺ and CD137⁺. Tetanus toxoid (TET) was used as a positive control in activation experiments. (B) PBMCs from BNT162b2 vaccinated HCWs (n=23), convalescent COVID-19 patients (n=15), and negative non-vaccinated controls (n=13) were stimulated with SARS-CoV-2 wild type spike protein peptide pool (0.5 µg/ml) for 48h. SARS-CoV-2 spike specific responses are indicated as stimulation indices (SI) calculated by dividing the percentage of AIM⁺ (CD69⁺CD134⁺) CD4⁺ and AIM⁺ (CD69⁺CD137⁺) CD8⁺ T cells with the percentage of AIM⁺ cells after DMSO stimulation. Results are from 20, 15, and 17 samples collected 6 weeks, 3 months, and 6 months after the first vaccine dose, respectively, and 11 negative controls Comparisons between the groups were performed with the Mann-Whitney U-test and paired analysis was done with the Wilcoxon signed rank test. ***p < 0.001; ****p < 0.0001. The differences between 3wk, 3mo and 6mo sample groups were statistically not significant.

Figure 3

T cell responses against SARS-CoV-2 variants of concern Alpha, Beta, Gamma and Delta. PBMCs collected from HCWs after BNT162b2 vaccination, COVID-19 patients, and negative controls were treated with DMSO or stimulated with peptide pools spanning the spike protein from SARS-CoV-2 wild type (S_wt) and four variants of concern Alpha, Beta, Gamma, and Delta. Stimulation indices (SI) were calculated by dividing the percentage of AIM⁺ cells after spike peptide pool stimulation with the percentage of AIM⁺ cells after DMSO stimulation. (A) CD4+ and (B) CD8+ T cell response was analyzed with the AIM assay and antigen specific CD4⁺ responses were defined with the expression of CD69⁺ and CD134⁺, and CD8⁺ responses with the expression of CD69⁺ and CD137⁺. Paired samples were analyzed with the Wilcoxon signed rank test and samples with no data on both data points were excluded from analysis. *p < 0.05.

Figure 4

Cytokine mRNA expression in PBMCs and secretion of cytokines after stimulation with SARS-CoV-2 spike-specific peptides. Cytokine analysis was performed for PBMCs collected from BNT162b2 vaccinated HCWs 6 weeks (n=15), 3 months (n=15), and 6 months (n=14) after the first vaccine dose and from convalescent COVID-19 patients (n=10) and negative controls (n=10). PBMCs were treated with DMSO or stimulated with tetanus toxoid (10 µg/ml), SARS-CoV-2 wt or Delta S protein peptide pools (0.5 µg/ml) for 48h. Delta variant S peptide stimulation was performed to 7/15 vaccinated, 6/10 COVID-19 patients, and 3/10 negative controls. Cells and supernatants were collected, and total cellular RNA was isolated from stimulated cells. (A) IFN-γ and IL-2 mRNA expression was quantitated from total cellular RNA with RT-qPCR and the data is presented as fold change in comparison to DMSO treated cells. (B) Secreted cytokines (IFN-γ and IL-2) were analyzed with multiplex immunoassay from PBMCs. IFN-γ measurement was unsuccessful from 1 6wk-sample, 1 3mo-sample, and 3 6mo-samples stimulated with DMSO or S_wt. IL-2 measurement was unsuccessful from 3 6wk-samples, 5 3mo and 6mo-samples, and 3 negative samples stimulated with DMSO or S_wt. Data is represented as median and interquartile range. Statistical analysis was performed by Wilcoxon signed rank test for comparison of wt S peptide pool stimulations with Delta S peptide pool stimulations (A, B) and tetanus and SARS-CoV-2 wt and Delta S peptide pool stimulations with DMSO control (B). Samples with no data on both data points were excluded from analysis. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Correlation of spike-specific T cell responses. Nonparametric Spearman correlation analysis of secreted IFN-γ and IL-2 concentrations with AIM⁺ CD4⁺ and CD8⁺ T cells in wild type (wt) SARS-CoV-2 spike protein-specific peptide pool stimulated PBMCs. Results are shown from 15 BNT162b2 vaccinated health care workers at three timepoints after two doses of BNT162b2 vaccine (n=37 fro IFNg and 32 for IL-2), 10 COVID-19 patients, and 8 for IFNG and 3 for IL-2 healthy non-vaccinated controls. Dotted lines indicate 95% CI.

Figure 6

Correlation of humoral immunity to cell-mediated immunity after BNT162b2 vaccination n = 23 and SARS-CoV-2 infection. (A) Anti-SARS-CoV-2 S1-specific IgG, S1 total Ig, and N protein-specific IgG antibody responses were measured from samples collected at 6 weeks, 3 months, and 6 months after vaccination n = 23 or 1 month after PCR confirmed SARS-CoV-2 infection (n=15). Serum antibody levels of vaccinated and infected individuals were compared with negative controls (n=13) that had not received any COVID-19 vaccines or suffered from a previous SARS-CoV-2 infection. Bars represent the geometric mean titers. The cut-off values for a positive test result are shown with dotted line Statistical analysis was performed with Mann Whitney U-test for comparison of 6wk vaccinee samples with COVID-19 patient samples. ****p<0.0001. (B,C) The correlation of anti-SARS-CoV-2 S1 IgG antibody levels with SARS-CoV-2 (wt) spike specific CD4⁺ and CD8⁺ T cell responses in (B) vaccinated HCWs (samples collected 6 week, 3 months, and 6 months after vaccination, n= 52) and (C) COVID-19 patients (samples collected 1 month post onset of symptoms, n=15). Correlation was analyzed with the Spearman’s correlation and Spearman’s r is indicated in the figures.