Breadth and Durability of SARS-CoV-2-Specific T Cell Responses following Long-Term Recovery from COVID-19

Abstract

T cell immunity is crucial for long-term immunological memory, but the profile of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific memory T cells in individuals who recovered from COVID-19 (COVID-19-convalescent individuals) is not sufficiently assessed. In this study, the breadth and magnitude of SARS-CoV-2-specific T cell responses were determined in COVID-19-convalescent individuals in Japan. Memory T cells against SARS-CoV-2 were detected in all convalescent individuals, and those with more severe disease exhibited a broader T cell response relative to cases with mild symptoms. Comprehensive screening of T cell responses at the peptide level was conducted for spike (S) and nucleocapsid (N) proteins, and regions frequently targeted by T cells were identified. Multiple regions in S and N proteins were targeted by memory T cells, with median numbers of target regions of 13 and 4, respectively. A maximum of 47 regions were recognized by memory T cells for an individual. These data indicate that SARS-CoV-2-convalescent individuals maintain a substantial breadth of memory T cells for at least several months following infection. Broader SARS-CoV-2-specific CD4⁺ T cell responses, relative to CD8⁺ T cell responses, were observed for the S but not the N protein, suggesting that antigen presentation is different between viral proteins. The binding affinity of predicted CD8⁺ T cell epitopes to HLA class I molecules in these regions was preserved for the Delta variant and at 94 to 96% for SARS-CoV-2 Omicron subvariants, suggesting that the amino acid changes in these variants do not have a major impact on antigen presentation to SARS-CoV-2-specific CD8⁺ T cells. IMPORTANCE RNA viruses, including SARS-CoV-2, evade host immune responses through mutations. As broader T cell responses against multiple viral proteins could minimize the impact of each single amino acid mutation, the breadth of memory T cells would be one essential parameter for effective protection. In this study, breadth of memory T cells to S and N proteins was assessed in COVID-19-convalescent individuals. While broad T cell responses were induced against both proteins, the ratio of N to S proteins for breadth of T cell responses was significantly higher in milder cases. The breadth of CD4⁺ and CD8⁺ T cell responses was also significantly different between S and N proteins, suggesting different contributions of N and S protein-specific T cells for COVID-19 control. Most CD8⁺ T cell epitopes in the immunodominant regions maintained their HLA binding to SARS-CoV-2 Omicron subvariants. Our study provides insights into understanding the protective efficacy of SARS-CoV-2-specific memory T cells against reinfection.

Keywords: COVID-19; SARS-CoV-2; T cell immunity; variants.

FIG 1

Magnitude of SARS-CoV-2-specific memory T cells in COVID-19-convalescent individuals. T cell responses against SARS-CoV-2 proteins in PBMCs (a to c) and in vitro expanded PBMCs (d to g) were measured by the IFN-γ ELISpot assay. Bars show the total T cell responses specific to all SARS-CoV-2 proteins tested for each individual. (a and d) Corrected (minus the negative control) SFC/10⁶ PBMCs. (b and f) Magnitude (SFC/10⁶ PBMCs) and frequency (percent) of T cell responses against each protein. (c) Magnitude (SFC/10⁶ PBMCs) of T cell responses specific for the indicated proteins between groups with mild and severe COVID-19. (e) Magnitude of T cell-specific responses to SARS-CoV-2 proteins before (PBMC) and after (Expanded) in vitro expansion. (g) Number of proteins recognized by memory T cells for the 11 SARS-CoV-2 proteins tested in the mild and the severe groups. Negative responses are shown as open circles in panels b, c, e, and f. Differences between groups were determined using the paired t test (ex vivo versus expanded PBMCs) and the Mann-Whitney test. ns, not significant.

FIG 2

Distribution of T cell-target regions in spike (S) and nucleocapsid (N) proteins. Frequency of responders (upper graphs) and magnitude of T cells response (lower graphs) to individual overlapping peptide (OLP) pairs in SARS-CoV-2 S and N proteins are shown. The OLP pairs that recognized more than 20% of tested individuals are shown as red bars. For graphs showing frequency of responders, the black line at the y axis indicates the cutoff for defining regions frequently targeted by T cells (20%). For graphs showing the magnitude of the ELISpot response, the black line on the y axis shows the cutoff for samples selected for ICS (≥300 SFC/million expanded PBMCs). NTD, N-terminal domain; RBD, receptor binding domain; FP, fusion peptide; CT, C terminus; CTD, C-terminal domain.

FIG 3

Frequent T cell target regions in spike and nucleocapsid proteins. aa, amino acid. *, overlapped peptide pairs are shown by shading. **, the numbers of the samples for which ICS was performed (the samples with ≥300 SFC/expanded PBMCs in the ELISpot assay) are shown. The frequencies of the responders with CD4⁺ and CD8⁺ T cell responses are shown as the intensities of blue and red, respectively.

FIG 4

Breadth of SARS-CoV-2-specific memory T cells. (a) Number of overlapping peptide (OLP) pairs that were recognized in S and N proteins by PBMCs expanded in vitro from COVID-19-convalescent individuals. (b) Comparison of the breadth of memory T cells in whole S and N proteins and the RBD between the mild and severe disease groups. (c) Correlation of the breadth and magnitude of memory T cells between S and N proteins. The ratio of N protein-specific to S protein-specific T cell responses (N/S) was calculated for both the breadth and the magnitude of the T cell response. Differences between groups were determined using the Mann-Whitney test. Correlations were performed using Spearman’s test.

FIG 5

CD4⁺ and CD8⁺ T cell responses against regions of SARS-CoV-2 spike and nucleocapsid proteins frequently targeted by T cells. (a) Representative flow cytometry plots of intracellular cytokine staining (ICS) from a single individual. The T cell response against each OLP pair was defined as positive when the frequency of IFN-γ⁺ TNF-α⁺ T cells was >0.1%. (b and c) CD4⁺ and CD8⁺ T cell responses for frequently targeted SARS-CoV-2 S and N regions. ICS was performed on OLP pairs using in vitro-expanded PBMCs with results of >300 SFC/million cells in the ELISpot assay. The bars show the median response. (d and e) Number of SARS-CoV-2 S and N regions targeted by CD4⁺ and CD8⁺ T cells for each individual assessed. Paired t test (d) and Mann-Whitney U test (e) were used for comparisons.