T cell responses after COVID-19 and vaccinations

Antibodies induced after vaccination against coronavirus disease 2019 (COVID-19) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have reduced ability to neutralize variants of concern (COVs). However, antibodies are not the only mediators of immunity.

A new study published in the journal Frontiers in immunology examines cell-mediated immunity in BNT162b2 mRNA COVID-19 vaccinated healthcare workers and COVID-19 patients.

Study: Durable T cell responses in BNT162b2 COVID-19 mRNA vaccinates and recovering COVID-19 patients. Image Credit: Corona Borealis Studio / Shutterstock

Cell-mediated immunity

T lymphocytes are the mediators of cellular immunity. They play an essential role in immune protection, recovery from acute infection and long-lasting immune memory.

Antigen-presenting cells (APCs) present several short peptides of viral proteins to T cells. T cell responses are stimulated by these different short peptides. Therefore, T cell responses are not sensitive to viral protein mutations like antibody responses.

CD8+ cytotoxic T lymphocytes (CTL) identify and destroy virus-infected cells. CD4+ T helper (Th) cells coordinate and enhance CTL responses. They also stimulate the production of antibodies by B cells.

Previous studies have shown that SARS-CoV-2 infection stimulates robust memory CD4+ and CD8+ T cell responses. This cell-mediated immunity can provide long-lasting protection against reinfections.

Cell-mediated immunity assessment

This study analyzes the longevity of SARS-CoV-2 spike-specific antibody and cell-mediated immunity in BNT162b2-vaccinated healthcare workers and COVID-19 patients.

This study included 23 fully vaccinated healthcare workers (two doses of BNT162b2 vaccine), 15 COVID-19 patients, and 13 people with no previous SARS-CoV-2 infection or COVID-19 vaccination. Blood samples were taken from vaccinees six weeks, three months, and six months after the first dose of vaccine; convalescent COVID-19 patients at 18 to 45 days (mean 33 days) after SARS-CoV-2 infection.

Peripheral blood mononuclear cells (PBMCs) were isolated from blood and activated with full-length SARS-CoV-2 spike protein peptides. T cell subtypes within the PBMC population were characterized by flow cytometry.

The expression of different cytokines was measured by RT-qPCR. Additionally, the levels of cytokines and other molecules secreted by the cells were detected using Luminex. Antibodies to SARS-CoV-2 were measured by enzyme immunoassay (EIA). The activation-induced marker (AIM) assay was optimized prior to sample analysis.

PBMCs were stimulated with the wild-type SARS-CoV-2 Wuhan virus spike peptide pool. 48h stimulation and measurement of IFN-γ and IL-2 mRNA levels were selected for all analyses. Tetanus toxoid was used as a positive control. IFN-γ and IL-2 are Th1 type cytokines.

T cell responses

SARS-CoV-2 spike-specific CD4+ cellular responses were detected in all COVID-19 patients and vaccinated individuals 6 weeks, 3 months and 6 months after the first vaccine dose.

CD8+ cellular responses were detected in 80% of COVID-19 patients and 70% of vaccinees 6 weeks after the first dose; in 67% of people vaccinated 3 months after the first dose; and in 53% of those vaccinated 6 months after the first dose. These T-cell responses were similar in COVID-19 patients and vaccinated people.

T cell responses against COVs were assessed by stimulating PBMCs with pools of peptides from spike proteins of Alpha, Beta, Gamma and Delta variants. CD4+ cell responses against all tested COVs were detected in more than 71% of vaccinees and more than 75% of COVID-19 patients.

CD8+ T cell responses against all COVs tested were detected in over 50% of vaccinees and over 75% of patients with COVID-19. However, significant differences were observed between wild type and Gamma variant 6 weeks after vaccination and wild type and Beta variant 6 months after vaccination.

COVID-19 patients had stronger CD4+ and CD8+ responses than vaccinated individuals. There was no decrease in cell-mediated immunity against VOCs after 6 months.

IFN-γ and IL-2 mRNA expression was detected in 93% and 100% of vaccinated individuals after stimulation with the wild-type and Delta variant spike peptide pool. mRNA levels were higher than mRNA levels in PBMCs taken from unvaccinated and uninfected individuals. IFN-γ and IL-2 mRNA expression was similar between COVID-19 patients and vaccinated individuals.

Correlation of humoral immunity to cell-mediated immunity after BNT162b2 n=23 vaccination 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 post-vaccination n=23 or 1 month after PCR confirmation of SARS-CoV-2 infection (n=15).  Serum antibody levels of vaccinated and infected individuals were compared to negative controls (n=13) who had received no COVID-19 vaccine or who had suffered from a previous SARS-CoV-2 infection.  Bars represent geometric mean titers.  Cut-off values ​​for a positive test result are indicated by a dotted line.  Statistical analysis was performed with Mann Whitney's U test for comparison of 6-week-old vaccinee samples with COVID-19 patient samples.  ****p<0,0001.  (B,C) La corrélation des niveaux d'anticorps anti-SARS-CoV-2 S1 IgG avec les réponses des lymphocytes T CD4+ et CD8+ spécifiques au pic SARS-CoV-2 (wt) chez (B) les travailleurs de la santé vaccinés (échantillons prélevés 6 semaines, 3 mois , et 6 mois après la vaccination,

Correlation of humoral immunity to cell-mediated immunity after BNT162b2 n=23 vaccination 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 post-vaccination n=23 or 1 month after PCR confirmation of SARS-CoV-2 infection (n=15). Serum antibody levels of vaccinated and infected individuals were compared to negative controls (n=13) who had received no COVID-19 vaccine or who had suffered from a previous SARS-CoV-2 infection. Bars represent geometric mean titers. Cut-off values ​​for a positive test result are indicated by a dotted line. Statistical analysis was performed with Mann Whitney’s U test for comparison of 6-week-old vaccinee samples with COVID-19 patient samples. ****p

After stimulation of PBMCs from vaccinated individuals with wild-type and Delta-variant spike peptide pools, IFN-γ and IL-2 protein levels increased significantly. IFN-γ and IL-2 levels were higher in vaccinated individuals and COVID-19 patients than in unvaccinated uninfected individuals. IFN-γ and IL-2 levels did not decrease 6 months after vaccination. Additionally, stimulation with wild-type or Delta-variant peptide pools produced equivalent levels of IFN-γ and IL-2.

There was a strong correlation between T cell activation and the production of IFN-γ and IL-2 cytokines.

All vaccinated individuals produced peak-specific anti-SARS-CoV-2 antibodies six weeks after the first vaccine dose. Antibody levels were higher than those of hospitalized COVID-19 patients. In vaccinated individuals, antibody levels gradually declined.

Anti-SARS-CoV-2 antibody levels in vaccinated individuals did not correlate with CD4+ or CD8+ T cell responses. However, in COVID-19 patients, spike-specific anti-SARS-CoV-2 antibodies increased with increasing CD4+ and CD8+ T cell responses.

Study limitations

This study cryopreserved PBMCs until analysis. This can decrease cell viability and some memory cells may be lost in the process. The number of participants assessed in this study was relatively small. Only individuals vaccinated with BNT162b2 were studied. This study did not include vaccinated people over the age of 60. The AIM protocol did not test longer incubation times. Longer 15-mer peptide pools were used for stimulation instead of 9-10-mer peptides. This may underestimate SARS-CoV-2 specific CD8+ cells.

Conclusion

This study shows that although antibodies specific to the SARS-CoV-2 spike protein decline over time after COVID-19 vaccination or natural infection, cell-mediated immunity is retained to a large extent and is not susceptible to mutations in the viral spike protein.

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