Long-lived memory T lymphocyte responses against SARS coronavirus nucleocapsid protein in SARS-recovered patients

Abstract

The nucleocapsid (N) protein is a structural component of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and can induce antibody responses in SARS patients during infection. However, it is not known whether SARS-CoV N protein can induce a long persistence of memory T-cell response in human. In this study, we found that peripheral blood mononuclear cells (PBMCs) from fully recovered SARS individuals rapidly produced IFN-gamma and IL-2 following stimulation with a pool of overlapping peptides that cover the entire N protein sequence. The N-specific IFN-gamma(+)CD4(+) T cells were mainly composed of CD45RA(-)CCR7(+)CD62L(-) cells, whereas IFN-gamma(+)CD8(+) memory T cells were mostly contained within CD45RA(+)CCR7(-)CD62L(-) cell population. Epitope mapping study indicated that a cluster of overlapping peptides located in the C-terminal region (amino acids [aa] 331 to 362) of N protein contained at least two different T-cell epitopes. The results indicated that human memory T-cell responses specific for SARS-CoV N protein could persist for 2 years in the absence of antigen, which would be a valuable for the design of effective vaccines against SARS-CoV and for basic studies of human T-cell memory.

Figures

Fig. 1

Production of IFN-γ by PBMCs from SARS-recovered individuals induced with a pool of SARS-CoV N peptides. (A) PBMCs from seven SARS-recovered donors were cultured in 96-well plates with or without a pool of N peptides for 72 h. The culture supernatants were collected and assessed for the production of IFN-γ by ELISA. All of assays were performed in triplicate. Statistical analysis used a Student's t test. Statistical significant difference was set at P < 0.05(**). *P > 0.05. Bars indicate mean values. (B) PBMCs from eleven SARS-recovered donors were stimulated with a pool of N peptides. IFN-γ-producing cells were detected by ELIspot assay. PBMCs incubated with medium alone were used as negative controls. The number of spots in the medium control wells ranged from 0 to 2. All of assays were performed in triplicate. Statistical analysis used a Student's t test. Statistical significant difference was set at P < 0.05(**). *P > 0.05. Bars represent mean values.

Fig. 2

Detection of IFN-γ-producing T cells specific to SARS-CoV N peptides by intracellular staining. PBMCs from SARS-recovered donors were incubated with a pool of peptides for 6 h. Cell surface and intracellular cytokine staining for IFN-γ was performed. The cells were first gated on CD4−CD8+ T cells (A) and CD8−CD4+ T cells (B) and analyzed for IFN-γ expression. Results shown are from one experiment performed on PBMC from a SARS patient and are representative of 5 independent experiments from 5 SARS patients. C represents the expression of IFN-γ as percentage of total CD4+ or CD8+ T cells in different patients. Bars indicate mean values.

Fig. 3

Cytokine profiles of IL-2 and IFN-γ in CD4+ and CD8+ T cells in response to N peptides. PBMCs from SARS-recovered donors were stimulated with a pool of N peptides for 6 h. The expression of IL-2 and IFN-γ was assessed by flow cytometry. (A) Dot plots show intracellular staining for IL-2 and IFN-γ in the CD8+ T cells and CD4+ T cells. Data are representative of three independent experiments with similar results. (B) The frequency of cells expressing both IL-2 and IFN-γ in PBMCs from SARS-recovered donors. Data are presented as the percentage of the total number of cells expressing at least one cytokine.

Fig. 4

Determination of phenotype of N-protein-specific memory T cells in SARS-recovered donors. PBMCs from SARS-recovered donors were stimulated with a pool of N peptides for 6 h. The expression of CD45RA, CCR7 and CD62L in IFN-γ+CD4+ or IFN-γ+CD8+ T cells was assessed by flow cytometry. CD4 +CD8− or CD4 +CD8− cells were gated and analyzed. Results shown are from one experiment performed on PBMC from a SARS patient and are representative of 3 independent experiments from 3 SARS patients.

Fig. 5

Specific T-cell responses to pooled peptides of the N protein in SARS-recovered donors by ELISA and ELIspot assay. Fifty seven overlapping individual peptides that covered the entire N protein sequence were divided into 6 pools (each pool contains 10 peptides) as described in Materials and methods. (A) PBMCs from 5 SARS-recovered donors were stimulated with a pool of 10 peptides (1 μg/ml of each peptide) for 72 h. The culture supernatants were collected for detection of IFN-γ by ELISA. Bars indicate mean values. (B) PBMCs from four SARS-recovered donors were stimulated with a pool of 10 peptides (1 μg/ml of each peptide) for detection of IFN-γ expression by ELIspot assay. PBMCs incubated with media alone were used as negative controls. The experiments were carried out in triplicate.

Fig. 6

T-cell responses to individual peptide of SARS-CoV N protein by ELIspot assay. PBMCs from SARS-recovered donors were stimulated with a single peptide (1 μg/ml). The production of IFN-γ was assessed by ELIspot assay. The number of spots in the media control wells was ranged from 0 to 2. The experiments were carried out in triplicate.

Fig. 7

Memory T-cell responses specific to the peptides N46, N47 or N48 by intracellular staining. PBMCs from SARS-recovered donors were incubated with the indicated peptides (N46, N47 or N48) for 6 h. The intracellular staining for IFN-γ was performed. CD4+ and CD8+ T cells were gated and analyzed for IFN-γ expression by flow cytometry (A). Results shown are representative of 3 independent experiments from 3 SARS patients. Similar results were also obtained in other experiments for CD8+ (B) and CD4+ (C) T cells.