Conceived and designed the experiments: ZC LZ TL JX YH LB. Performed the experiments: ZC LZ QF TL JX YH HF ZQ YH WX WZ HY YZ JY. Analyzed the data: ZC LZ QF TL JX HF LB ZQ YH WX WZ HY YZ JY. Contributed reagents/materials/analysis tools: LZ. Wrote the paper: LZ
The authors have declared that no competing interests exist.
Most of the individuals infected with SARS coronavirus (SARS-CoV) spontaneously recovered without clinical intervention. However, the immunological correlates associated with patients' recovery are currently unknown. In this report, we have sequentially monitored 30 recovered patients over a two-year period to characterize temporal changes in SARS-CoV-specific antibody responses as well as cytotoxic T cell (CTL) responses. We have found persistence of robust antibody and CTL responses in all of the study subjects throughout the study period, with a moderate decline one year after the onset of symptoms. We have also identified two potential major CTL epitopes in N proteins based on ELISPOT analysis of pooled peptides. However, despite the potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients have not yet been restored to normal levels. In summary, our study has, for the first time, characterized the temporal and dynamic changes of humoral and CTL responses in the natural history of SARS-recovered individuals, and strongly supports the notion that high and sustainable levels of immune responses correlate strongly with the disease outcome. Our findings have direct implications for future design and development of effective therapeutic agents and vaccines against SARS-CoV infection.
SARS, or severe acute respiratory syndrome, is a serious respiratory illness caused by a novel variant of coronavirus (SARS-associated coronavirus, SARS-CoV)
Starting in March 2003, we have enrolled and sequentially followed up 30 patients who were diagnosed and recovered from SARS-CoV infection according to clinical criteria released by the World Health Organization (
Using flow cytometry, we first studied the sequential changes in the absolute numbers of total lymphocytes, CD3, CD4, CD8 T lymphocytes, B lymphocytes and natural killer (NK) cells over the two years follow-ups and compared with that from normal healthy controls. As show in
Grey stars indicate the significant differences between the recovered patients at 24 months after onset of symptom and normal healthy controls.
Their respective
To study the sequential changes in humoral responses against SARS-CoV, we used our previously published ELISA-based and pseudotyped retrovirus-based neutralization systems
Serum samples were diluted 100- and 900-fold prior to ELISA and neutralization studies.
Both dilutions were used to carried out experiments presented in (B) and (C), where the only 100-fold diluted serum were used for experiments presented in (A).
The top and bottom of each rectangular box denote the 75th and 25th percentiles, respectively, with the median shown inside the box.
Horizontal bars extending from each box represent the 90th and 10th percentiles.
Significant differences between samples are indicated by grey stars with their respective
Otherwise no significance was found.
To study the sequential changes in CTL responses against SARS-CoV, we used ELISPOT-based technique to quantify the number of INF-γ releasing cells in the peripheral blood against peptide pools covering the entire N protein derived from the Urbani strain
(A) The average number of spot forming cells (SFC) per million of PBMC from recovered patients at month 3, 12, and 18 post onset of symptom.
No significant differences were found among these samples.
The top and bottom of each rectangular box denote the 75th and 25th percentiles, respectively, with the median shown inside the box.
Horizontal bars extending from each box represent the 90th and 10th percentiles.
(B) The average number of SFC per million of PBMC against various pools of N protein peptides (open rectangle) and percent of PBMC samples recognizing various pools of N protein peptides (closed rectangle).
An arbitrary line was draw at 3000 SFC per million PBMC to identify peptide pools that are preferentially recognized by the recovered patients in this cohort.
The 57 peptides (one is not listed) were pooled into 15 groups, 7 of which (NX1-7) are listed vertically and 8 are (NY1-8) listed horizontally.
The actual amino acid residue sequences preferentially recognized by the recovered patients are highlighted (lower panel), which correspond to residue sequences between position number 211 to 235 and 330 to 354.
A | Frequency | B | Frequency | DR | Frequency | DQ | Frequency |
A1 | 2.08 | B7 | 4.35 | DR1 | 6.38 | DQ2 | 4.08 |
A2 | 27.08 | B13 | 6.52 | DR4 | 12.77 | DQ5 | 16.33 |
A3 | 14.58 | B27 | 2.17 | DR7 | 4.26 | DQ6 | 22.45 |
A11 | 25.00 | B35 | 8.70 | DR8 | 6.38 | DQ7 | 24.49 |
A24 | 12.50 | B37 | 2.17 | DR9 | 17.02 | DQ8 | 12.24 |
A26 | 2.08 | B46 | 8.70 | DR10 | 2.13 | DQ9 | 18.37 |
A29 | 2.08 | B49 | 4.35 | DR11 | 8.51 | ||
A30 | 4.17 | B51 | 10.87 | DR12 | 17.02 | ||
A31 | 2.08 | B52 | 2.17 | DR13 | 4.26 | ||
A33 | 6.25 | B54 | 2.17 | DR14 | 2.13 | ||
A203 | 2.08 | B58 | 6.52 | DR15 | 12.77 | ||
B60 | 15.22 | DR16 | 4.26 | ||||
B61 | 4.35 | DR17 | 2.13 | ||||
B62 | 8.70 | ||||||
B67 | 2.17 | ||||||
B75 | 4.35 | ||||||
B81 | 2.17 | ||||||
B3901 | 2.17 | ||||||
B5102 | 2.17 |
In this report, we have extended our early study by sequentially monitoring 30 recovered SARS patients over a 2-year period to characterize temporal changes in humoral and CTL responses against SARS-CoV. We have shown for the first time that recovered patients have persistent and robust binding as well as neutralizing antibody and CTL responses throughout the study period with a moderate decline one year after the onset of symptoms. In particular, S glycoprotein-specific Nab responses are persistently high in the recovered patients even 24 months after onset of symptom. Such high and sustainable levels of SARS-CoV-specific immune responses in these patients are clear distinction from that found in patients who succumbed to the disease
Thirty SARS patients were enrolled in March 2003 and have continuously followed up since then. They were diagnosed and recovered from SARS-CoV infection according to clinical criteria released by the World Health Organization (
For flow cytometric analyses of various lymphocyte populations in the peripheral blood, we used our previously published protocols
Analysis of binding antibodies against whole SARS-CoV lysates or N protein, and S glycoprotein-specific neutralizing antibody (Nab) were conducted as previously reported
ELISPOT assays were performed using a commercially available kit (Diaclone, France) according to manufacture's introduction. The peptide pools covering the entire N protein of the Urbani strain were used to stimulate patients' peripheral blood mononuclear cells. The peptide pools were made of 57 peptides which are 15–18 amino acid residues in length overlapping by 10 amino acid residues. These peptides were obtained through the Biodefense and Emerging Infections Resources Repository at National Institute of Health (NIH) in the United States (
Student's
We feel in debt to patients' willingness to participate our study.