T cell and antibody kinetics delineate SARS-CoV-2 peptides mediating long-term immune responses in COVID-19 convalescent individuals

Abstract

Long-term immunological memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for the development of population-level immunity, which is the aim of vaccination approaches. Reports on rapidly decreasing antibody titers have led to questions regarding the efficacy of humoral immunity alone. The relevance of T cell memory after coronavirus disease 2019 (COVID-19) remains unclear. Here, we investigated SARS-CoV-2 antibody and T cell responses in matched samples of COVID-19 convalescent individuals up to 6 months after infection. Longitudinal analysis revealed decreasing and stable spike- and nucleocapsid-specific antibody responses, respectively. In contrast, functional T cell responses remained robust, and even increased, in both frequency and intensity. Single peptide mapping of T cell diversity over time identified open reading frame-independent, dominant T cell epitopes mediating long-term SARS-CoV-2 T cell responses. Identification of these epitopes may be fundamental for COVID-19 vaccine design.

Fig. 1. Longitudinal clinical and immunological analysis of convalescent donors after SARS-CoV-2 infection.

(A) Prevalence, quantity and character of post-infectious symptoms in convalescent COVID-19 donors (total n = 51, symptomatic n = 14) at T2. (B) Schematic overview of the experimental workflow for the longitudinal analysis of immune responses in convalescent donors (n = 51). Intensity of T cell responses was assessed in group A (n = 29) using SARS-CoV-2-specific and cross-reactive epitope compositions (EC) comprising multiple HLA class I- and HLA-DR-restricted SARS-CoV-2-specific and cross-reactive T cell epitopes in ex vivo IFN-γ ELISPOT assays. Diversity of T cell responses was analyzed in group B (n = 23) by single peptide screening using 20 HLA-DR-restricted and 6 HLA-A*24-restricted SARS-CoV-2-derived peptides. (C and D) Proportion of convalescent donors with T cell responses to SARS-CoV-2-specific (C) and cross-reactive (D) EC at T1 and T2.

Fig. 2. Longitudinal analysis of SARS-CoV-2 T cell response intensity in convalescent individuals.

(A) A representative example of T cell responses to HLA class I- and HLA-DR-restricted SARS-CoV-2-specific and cross-reactive epitope compositions (EC) assessed by ex vivo IFN-γ ELISPOT assays at T1 and T2 in using cells isolated from the convalescent donor UDN121. Data are presented as scatter dot plot with bars indicating the mean spot counts of duplicates normalized to 5 × 10⁵ cells. UDN, uniform donor number; EC, epitope compositions; Spec EC, SARS-CoV-2-specific EC; Cross EC, cross-reactive EC; Neg, negative control. (B and C) Intensities of ex vivo T cell responses to SARS-CoV-2-specific (B; HLA class I-restricted, n = 21; HLA-DR-restricted, n = 29) or cross-reactive (C; HLA class I-restricted, n = 11; HLA-DR-restricted, n = 29) EC at T1 and T2. Dots represent individual donors with detectable T cell response. Data were displayed as box plots (upper row) and line plots (lower row). P values were calculated using a Wilcoxon signed rank test. (D and E) Waterfall plots show ΔT2-T1 of T cell response intensity to HLA class I-restricted (D) and HLA-DR-restricted (E) SARS-CoV-2-specific EC. Donors with post-infectious symptoms are marked in orange. (F and G) CD4⁺ and CD8⁺ T cell responses at T2 against specific (F) and cross-reactive EC (G) were evaluated by flow cytometry. (H and I) Flow cytometry-based ex vivo characterization of cytokine profiles (IFN-γ, TNF, IL-2) and degranulation marker (CD107a) for CD8⁺ and CD4⁺ T cell responses at T2 against SARS-CoV-2-specific (H) and cross-reactive EC (I). Percentage of samples with CD107a⁺, IL-2⁺, TNF⁺, and IFN-γ⁺ SARS-CoV-2 T cell responses are shown in the upper rows. The lower rows display proportion of samples revealing mono- (1), di- (2), tri- (3), or tetra-functional (4) T cell responses.

Fig. 3. Dynamics of SARS-CoV-2 antibodies in relation to T cell responses and post-infectious clinical status.

(A to C) Antibody responses in convalescent donors (n = 51) at T1 and T2 for anti-S1 IgG (A) and IgA (B) or anti-nucleocapsid (C) antibodies. Donors with negative or borderline responses are marked in white or grey, respectively. Box plots are shown. P values were calculated using a Wilcoxon signed rank test. Ab, antibody. (D to F) Waterfall plots show change of anti-S1 IgG (D) and IgA (E) ratios or anti-nucleocapsid antibody titers (F) from T1 to T2 (Δ = T2-T1). Donors with post-infectious symptoms are marked in orange. (G) Heatmap of COVID-19 symptom scores (SC) and post-infectious symptoms, intensities of T cell responses to different EC (color gradient green) and antibody responses (color gradient purple) at T1 and T2 in individual donors (group A, n = 29). UDN, uniform donor number; Sym, symptoms; Spec, SARS-CoV-2-specific EC; Cross, cross-reactive EC; α-nuc, anti-nucleocapsid; α-spi, anti-spike; *, donors with borderline response. (H) Anti-S1 IgG and IgA ratios and anti-nucleocapsid titers at T2. (I) ΔT2-T1 of respective antibody responses. (J) Intensity of T cell responses at T2 and (K) ΔT2-T1 of intensity to SARS-CoV-2-specific EC restricted to HLA class I (n = 21) or HLA-DR (n = 29) and cross-reactive EC restricted to HLA class I (n = 11) or HLA-DR (n = 29). Data presented as box plots. P values were calculated using Mann-Whitney U tests.

Fig. 4. Longitudinal assessment of HLA-DR-directed T cell response diversity in convalescent donors.

(A) Diversity of T cell responses, which refer to the percentage of recognized HLA-DR-restricted peptides per donor (n = 18; group B), at T1 and T2 as analyzed by IFN-γ ELISPOT assays after 12-day in vitro pre-stimulation. Dots represent individual donors. Data were displayed as box plots (upper row) and line plots (lower row). P value was calculated using a Wilcoxon signed rank test. (B) Heatmap indicating positive (green) and negative (grey) T cell responses, as well as the intensities of T cell responses (color gradient green) to 20 HLA-DR-restricted SARS-CoV-2-derived peptides (DR_P01 - DR_P20) in individual donors (n = 18) at T1 and T2. (C) Recognition frequencies of peptides at T1 and T2 (top) and their variation over time (bottom, ΔT2-T1) grouped into dominant and subdominant peptides capable (persistent) or incapable (decreasing) of mediating persisting T cell response over time. ORF, open reading frame; nuc, nucleocapsid; spi, spike protein; env, envelope protein; mem, membrane protein.