Time-dependent contraction of the SARS-CoV-2-specific T-cell responses in convalescent individuals

Abstract

Background: Adaptive immunity in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is decisive for disease control. Delayed activation of T cells is associated with a worse outcome in coronavirus disease 2019 (COVID-19). Although convalescent individuals exhibit solid T-cell immunity, to date, long-term immunity to SARS-CoV-2 is still under investigation.

Objectives: We aimed to characterize the specific T-cell response on the basis of the in vitro recall of IFN-γ-producing cells to in silico-predicted peptides in samples from SARS-CoV-2 convalescent individuals.

Methods: The sequence of the SARS-CoV-2 genome was screened, leading to the identification of specific and promiscuous peptides predicted to be recognized by CD4⁺ and CD8⁺ T cells. Next, we performed an in vitro recall of specific T cells from PBMC samples from the participants. The results were analyzed according to clinical features of the cohort and HLA diversity.

Results: Our results indicated heterogeneous T-cell responsiveness among the participants. Compared with patients who exhibited mild symptoms, hospitalized patients had a significantly higher magnitude of response. In addition, male and older patients showed a lower number of IFN-γ-producing cells. Analysis of samples collected after 180 days revealed a reduction in the number of specific circulating IFN-γ-producing T cells, suggesting decreased immunity against viral peptides.

Conclusion: Our data are evidence that in silico-predicted peptides are highly recognized by T cells from convalescent individuals, suggesting a possible application for vaccine design. However, the number of specific T cells decreases 180 days after infection, which might be associated with reduced protection against reinfection over time.

Keywords: AIM, Activation-induced marker; COVID-19; COVID-19, Coronavirus disease 2019; DMSO, Dimethyl sulfoxide; ELISPOT, Enzyme-linked immunospot; OR, Odds ratio; SARS-CoV-2; SARS-CoV-2, Severe acute respiratory syndrome coronavirus-2; SFU, Spot-forming unit; T lymphocyte; VOC, Variant of concern; adaptive immunity.

Fig 1

Overall magnitude of T-cell response to the predicted SARS-CoV-2 peptides. Each column represents the sum of IFN-γ–producing T cells (SFU/10⁶) of the 121 samples in response to the individual peptides from SARS-CoV-2. A, Specific CD4⁺ T-cell response. B, Specific CD8⁺ T-cell response. E, Envelope protein; Ex, exonuclease protein; M, membrane protein; N3, nonstructural protein (NSP) 3; N6, NSP6; N9, NSP9; NC, nucleocapside protein; O1, open reading frame (ORF) 1; O3a, ORF3a; O7, ORF7; O8, ORF8; RP, RNA polymerase.

Fig 2

Magnitude of the specific IFN-γ production based on clinical characteristics. Each colunm represents the summed mean of the specific IFN-γ production (SFU × 10⁶) of the study participants in response to each peptide, grouped according to clinical charatcteristics as COVID-19 outcome, age, and sex. A-C, Specific CD4⁺ T-cell response. D-F, Specific CD8⁺ T-cell response. The relationship between categoric variables was analyzed by using the chi-square test. ∗∗∗∗P < .0001.

Fig 3

Long-term immunity to SARS-CoV-2 peptides. PMBC samples collected at 2 different time points (30 and 180 days after symptom onset) were stimulated with megapools containing 20 CD4 peptides (MCD4) and 26 CD8 peptides (MCD8). A, T-cell recall (IFN-γ production) 180 days after sympton onset. B and C, Paired analysis of the specific CD4⁺ and CD8⁺ T-cell IFN-γ production after 30 and 180 days, respectively. D and E, Overall fold of response decrease after 180 days according to clinical charatecteristics of the cohort. Matched-pair analysis was performed with the Wilcoxon test. ∗∗∗∗P < .0001.

Fig 4

Expression of AIMs in CD4⁺ and CD8⁺ T cells. The remaining PMBC samples collected at 2 different time points (30 and 180 days after sympton onset) were stimulated for 24 hours with 3 pools of peptides (peptides found in spike protein only, peptides found outside the spike protein, or a megapool containing all peptides [MCD4]). A, Gating strategy for CD4⁺ AIM⁺ T cells. B-D, CD4⁺ AIM⁺ T cells stimulated with peptides from spike protein, outside the spike protein, and MCD4, respectively. E, Gating strategy for CD8⁺ AIM⁺ T cells. CD8⁺ AIM⁺ T cells stimulated with peptides from spike protein, outside the spike protein, and MCD8, respectively.