HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Yang, L. Articles by Wu, C. Search for Related Content PubMed PubMed Citation Articles by Yang, L. Articles by Wu, C. Agricola Articles by Yang, L. Articles by Wu, C.

Persistent memory CD4⁺ and CD8⁺ T-cell responses in recovered severe acute respiratory syndrome (SARS) patients to SARS coronavirus M antigen

¹ Department of Immunology, Zhongshan Medical School, Sun Yat-sen University, Guangzhou 510089, China
² Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China
³ Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, China
⁴ Vaccine Research Center, NIAID/NIH, Bethesda, MD 20892, USA

Correspondence
Changyou Wu
changyou_wu{at}yahoo.com

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
The membrane (M) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is a major glycoprotein with multiple biological functions. In this study, we found that memory T cells against M protein were persistent in recovered SARS patients by detecting gamma interferon (IFN-) production using ELISA and ELISpot assays. Flow cytometric analysis showed that both CD4⁺ and CD8⁺ T cells were involved in cellular responses to SARS-CoV M antigen. Furthermore, memory CD8⁺ T cells displayed an effector memory cell phenotype expressing CD45RO^– CCR7^– CD62L^–. In contrast, the majority of IFN-⁺ CD4⁺ T cells were central memory cells with the expression of CD45RO⁺ CCR7⁺ CD62L^–. The epitope screening from 30 synthetic overlapping peptides that cover the entire SARS-CoV M protein identified four human T-cell immunodominant peptides, p21-44, p65-91, p117-140 and p200-220. All four immunodominant peptides could elicit cellular immunity with a predominance of CD8⁺ T-cell response. This data may have important implication for developing SARS vaccines.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that led to thousands of infected patients and hundreds of deaths. The aetiological agent of the syndrome, a novel coronavirus termed SARS coronavirus (SARS-CoV), has been identified (Drosten et al., 2003; Ksiazek et al., 2003). Although the epidemic was eventually controlled, the high morbidity and mortality associated with SARS make it imperative to develop effective vaccines to prevent this disease.

The immune system plays a vital role in defence against SARS-CoV infection, since none of the drugs employed to treat SARS is able to inhibit viral replication in vivo. Strong humoral responses have been found in most SARS patients following SARS-CoV infection, with high titres of neutralizing antibodies (Abs) present in their convalescent sera (Li et al., 2003). Sera from recovered SARS patients could be used to treat newly infected individuals (Soo et al., 2004), and passive transfer immune serum can prevent replication of SARS-CoV in a mouse model (Subbarao et al., 2004), confirming the protective nature of the anti-SARS-CoV serum Abs. UV-inactivated SARS-CoV vaccine was able to elicit a high titre of antibody responses in mice (Takasuka et al., 2004). Recent studies have demonstrated that vaccines based on the S protein of SARS-CoV seem to induce a considerable neutralizing antibody response and protective immunity (Yang et al., 2004; Bisht et al., 2004; Bukreyev et al., 2004). In addition to humoral response, T cells are essential for adaptive immunity against viral infection. Antiviral CD4⁺ T cells help the production of virus-specific Abs by B cells, while CD8⁺ cytotoxic T lymphocytes (CTLs) can kill virus-infected host cells.

The genome of SARS-CoV encodes four major structural components including the spike protein (S), the nucleocapsid protein (N), the envelop protein (E) and the membrane protein (M). To date, more attention has been focused on the antibody response and cellular immunity induced by the S and N proteins (Yang et al., 2004; Bisht et al., 2004; Bukreyev et al., 2004; Gao et al., 2003). Many studies suggest that the S protein of SARS-CoV is a promising antigen for developing SARS vaccines since it can elicit protective humoral immunity (Yang et al., 2004; Bisht et al., 2004) and that the N protein may serve as an ideal antigen for SARS diagnosis since it can induce an appreciable antibody response in SARS patients (Liu et al., 2004). In addition, human leukocyte antigen (HLA)-A2-restricted CD8⁺ T-cell epitopes have been identified in the S protein (Wang et al., 2004a, b; Zhi et al., 2005).We have demonstrated recently that memory CD4⁺ and CD8⁺ T cells specific for SARS-CoV S, E and N antigens from recovered SARS patients were persistent for a long period of time (Yang et al., 2006; Peng et al. 2006a, b). However, limited information is available on the immune response to the M protein of SARS-CoV.

The M protein of SARS-CoV is a major transmembrane glycoprotein of 220 aa. The interaction between the M protein and the S protein, as well as the N protein, is essential for viral assembly and budding. Recently, antibodies to the M protein that were detectable in convalescent-phase SARS patients and B-cell epitopes of the M protein have been identified (He et al., 2005). Although it has been demonstrated that DNA vaccine encoding the M protein of SARS-CoV can elicit antigen-specific T-cell responses in animal models (Jin et al., 2005; Wang et al., 2005), the protective effect of T cells directed to the M protein in humans remains undefined. Thus, it is essential to determine whether recovered SARS patients generate cell-mediated immunity against SARS-CoV and how long this immunity will persist. In this study, we analysed CD4⁺ and CD8⁺ T-cell responses to a pool of 30 overlapping SARS-CoV M peptides in recovered SARS patients, and characterized the phenotype of memory T-cell subpopulations, as well as sought to identify the immunodominant T-cell peptides on the M protein of SARS-CoV. Our study demonstrated that SARS-CoV M-specific memory CD4⁺ and CD8⁺ T cells were persistent in the peripheral blood of recovered SARS patients more than 1 year after infection. These memory T cells revealed effector and central memory phenotypes. Additionally, four human T-cell immunodominant peptides from the M protein of SARS-CoV were identified by eliciting gamma interferon (IFN-) production.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Donors.
Six fully recovered SARS patients, comprising four males and two females aged 25 to 32 years, worked in the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. All were infected with SARS-CoV between March and April 2003, these studies were performed 12–23 months after recovery. Four healthy donors, comprising two males and two females, whose ages ranged from 27 to 33 years, without any contact history with SARS-CoV, acted as healthy controls.

Synthetic peptides.
A total of 30 peptides, spanning the entire sequence of SARS-CoV M protein, were kindly provided by the Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, USA. The peptides range from 15 to 18 aa in length overlapped by 10–11 aa.

Reagents.
Anti-CD4 PerCP (peridinin chlorophyll protein) and anti-CD8 PerCP were purchased from BD Biosciences Immunocytometry Systems. Mouse anti-human CD28, anti-human CD49d, anti-CD4 FITC, anti-CD8 PE-Cy7 (cyanin 7), anti-CD45RO FITC, anti-CCR7 PE (phycoerythrin), anti-CD62L PE, anti-IFN- PE, anti-IFN- APC (allophycocyanin), anti-interleukin (IL)-2 APC and isotype-matched control antibodies were obtained from BD Bioscience Pharmingen. RPMI 1640 medium, penicillin, streptomycin and 2-mercaptoethanol were purchased from GIBCO.

Cell preparation.
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood that was mixed with an equal volume of Hank's balanced salt solution using Ficoll-Hypaque density gradient centrifugation. The cells were washed twice in RPMI 1640 medium supplemented with 10 % heat-inactivated fetal calf serum (FCS) (HyClone), 100 U penicillin ml^–1, 100 µg streptomycin ml^–1 and 50 µM 2-mercaptoethanol. The cells were adjusted to a final concentration of 2x10⁶ ml^–1 in complete RPMI 1640 medium.

Cell culture conditions and IFN- detection by ELISA.
Fresh PBMCs were isolated from recovered SARS patients and healthy donors, suspended in complete RPMI 1640 culture medium and stimulated with a pool of 30 overlapping SARS-CoV M peptides (1 µg ml^–1) in the presence of 1 µg anti-CD28 ml^–1 and 1 µg anti-CD49d ml^–1 mAbs. The final concentration of each peptide was 1 µg ml^–1. PBMCs were plated into a flat-bottomed 96-well plate at a total of 2x10⁵ cells per well and incubated for 72 h at 37 °C with 5 % CO₂ in triplicate. The supernatants were harvested and assayed for IFN- production by ELISA according to the manufacturer's protocol (BD Pharmingen). In each experiment, PBMCs were cultured with 1 µg anti-CD28 ml^–1 plus 1 µg anti-CD49d ml^–1 in the absence of SARS-CoV M peptides as a negative control. In addition, the cells were stimulated with 20 ng ml^–1 phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich) and 1 µg ionomycin ml^–1 (Sigma-Aldrich) as a positive control.

IFN- ELISpot assay.
Human IFN- ELISpot assays were performed using a commercially available set (BD Pharmingen) according to the manufacturer's instructions. Briefly, 96-well nitrocellulose plates (Millipore) were coated overnight at 4 °C with 5 µg ml^–1 anti-human IFN- capture antibody. The wells were then washed and blocked for 2 h at room temperature with 10 % FCS-RPMI 1640 medium. A total of 2x10⁵ PBMCs were added to each well and incubated for 20 h with a pool of SARS-CoV M peptides (1 µg ml^–1), 1 µg anti-CD28 ml^–1 and 1 µg anti-CD49d ml^–1 in triplicate. Similarly, the cells supplemented with anti-CD28 (1 µg ml^–1) and anti-CD49d (1 µg ml^–1) in the absence of SARS-CoV M peptides were considered as negative controls, and the cells were cultured with polyclonal stimuli (20 ng PMA ml^–1 plus 1 µg ionomycin ml^–1) as positive controls. The plates were washed and subsequently incubated with 2 µg ml^–1 biotinylated anti-human IFN- detection antibody for 2 h at room temperature. After washing, the wells were developed for 1 h with streptavidin-HRP, followed by addition of 3-amino-9-ethylcarbazole (AEC) substrate reagent. The spots were counted automatically using an ImmunoSpot image analyser (Cellular Technology). A spot represents an IFN--producing cell. The average of spots in triplicate wells was calculated and considered as the number of specific spot-forming cells (SFC) per 2x10⁵ cultured PBMCs.

Cell surface, intracellular cytokine staining and flow cytometric analysis.
PBMCs were stimulated with or without a mixture of 30 overlapping SARS-CoV M peptides in the presence of 1 µg anti-CD28 ml^–1 and 1 µg anti-CD49d ml^–1 in 15 ml tubes. After the first 1 h incubation, 10 µg brefeldin A ml^–1 (Sigma-Aldrich) was added to cultures to enable intracellular protein to accumulate in all stimulations. After incubation for a total of 5 h, the cells were washed, fixed, permeabilized using saponin (Sigma-Aldrich) and blocked with 25 µg human IgG ml^–1 for 30 min at 4 °C. The cells were then stained with anti-CD4, anti-CD8, anti-IFN-, anti-IL-2, anti-CD45RO, anti-CCR7 and anti-CD62L. After staining, all samples were washed twice in PBS buffer (containing 0.1 % saponin, 0.1 % BSA and 0.05 % NaN₃) and resuspended in 300 µl PBS for flow cytometry. More than 600 000 cells were acquired on a FACS Aria flow cytometer (Becton Dickinson) and FACS data were analysed using the CellQuest software (Becton Dickinson).

Statistical analysis.
The levels of IFN- and numbers of IFN--producing cells were compared using Student's t-test between recovered SARS patients and healthy control donors in the same condition. P<0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Quantification of SARS-CoV M peptides-specific memory T cells
To determine whether recovered SARS patients could maintain memory T-cell responses to the M protein of SARS-CoV more than 1 year after SARS-CoV infection, fresh PBMCs were isolated from fully recovered SARS patients and healthy donors and cultured with or without a pool of 30 SARS-CoV M peptides in the presence of anti-CD28 and anti-CD49d mAbs. After stimulation for 72 h, cell-free culture supernatants were harvested and assayed for the production of IFN- by ELISA. The results showed that PBMCs from healthy donors secreted very low levels of IFN- (0–78 pg ml^–1) when cultured with or without the SARS-CoV M peptides. Statistical analysis indicated that the production of IFN- between the two culture conditions was not significantly different (P>0.05) (Fig. 1a). In contrast, PBMCs from recovered SARS individuals produced significantly higher levels of IFN- (440±219 pg ml^–1) when stimulated with SARS-CoV M peptides (P<0.01) compared with the cells stimulated without SARS-CoV M peptides (50±31 pg ml^–1) (Fig. 1a). In addition, no marked difference in the production of IFN- between recovered SARS patients and healthy control individuals was observed when the PBMCs were stimulated with PMA plus ionomycin (P>0.05) (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 1. SARS-CoV M peptides-specific IFN- production by PBMCs. Fresh PBMCs were isolated from fully recovered SARS patients (Patients) and healthy control individuals (Controls), and cultured with or without a pool of 30 SARS-CoV M peptides (1 µg ml–1) in the presence of anti-CD28 (1 µg ml–1) and anti-CD49d mAbs (1 µg ml–1). After incubation for 72 h, the level of IFN- in culture supernatants was assayed by ELISA (a). The frequency of IFN--secreting cells was detected by ELISpot assay at the single cell level after stimulation for 20 h (b). The individual data points for each patient are means for triplicates. Student's t-test was used for statistical analysis.***, P<0.01. NS, not significant.

Subpopulation of SARS-CoV M peptides-specific memory T cells
It is well known that cellular immune responses are mediated by CD4⁺ and CD8⁺ T cells. We next analysed IFN--producing T-cell subpopulations that were involved in anti-SARS-CoV immunity by flow cytometry. PBMCs isolated from recovered SARS patients were stimulated with a pool of SARS-CoV M peptides (1 µg ml^–1) and co-stimulatory mAbs (1 µg anti-CD28 ml^–1 and 1 µg anti-CD49d ml^–1), in the presence of 10 µg brefeldin A ml^–1. After stimulation for 5 h, the cells were stained for surface expression of CD4 and CD8 and for intracellular IFN- and IL-2, or their corresponding isotype controls. The results showed that addition of SARS-CoV M peptides to cultures resulted in the expression of IFN- and IL-2 in both CD4⁺ (Fig. 2a) and CD8⁺ T-cell populations (Fig. 2b). In contrast, CD4⁺ and CD8⁺ T cells failed to secret IFN- and IL-2 in the absence of SARS-CoV M peptides. In addition, the percentage of IFN-- and IL-2-producing CD4⁺ and CD8⁺ T cells was significantly higher in SARS-CoV M peptides-stimulated cells compared with that from un-stimulated cells (P<0.05) (Fig. 2c), whereas no difference was observed in the proportion of IL-2-producing CD8⁺ T cells between those two groups (P>0.05). Based on the production of these two cytokines (IFN- and IL-2), SARS-CoV M peptides-specific CD4⁺ T cells could be divided into three distinct populations: IFN-⁺ IL-2^–, IFN-⁺ IL-2⁺ and IFN-^– IL-2⁺ (Fig. 2d). Overall, these data demonstrated that both CD4⁺ and CD8⁺ memory T cells were generated and persisted more than 1 year after SARS-CoV infection, and that these cells displayed a heterogeneity of cytokine expression.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Expression of intracellular IFN- and IL-2 in PBMCs of recovered SARS patients after stimulation with SARS-CoV M peptides in vitro. Fresh PBMCs from recovered SARS patients were cultured for 5 h with a mixture of 30 SARS-CoV M peptides (1 µg ml–1) and stained with anti-CD4, CD8, IFN- and IL-2 mAbs. The expression of IFN- and IL-2 by CD4+ (a) and CD8+ (b) T cells was analysed. The percentage (mean ±SD) of IFN-- or IL-2-secreting CD4+ and CD8+ T cells from six recovered SARS individuals is shown (c) and the subsets of M peptides-specific CD4+ T cells based on the expression of IFN- and IL-2 (d) are also displayed. The numbers in each quadrant are indicated as percentages. Representative data from one out of six recovered SARS individuals with similar results are shown (d). Student's t-test was used for statistical analysis. *, P<0.05; NS, not significant.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Phenotypic characteristics of SARS-CoV M peptides-specific memory CD4+ or CD8+ T cells. Blood mononuclear cells were stained with antibodies specific for CD4 or CD8, CD45RO, CCR7 and CD62L after stimulation for 5 h with a pool of 30 SARS-CoV M peptides (1 µg ml–1). The CD45RO, CCR7 and CD62L expression gated on either total CD4+ T cells or CD4+ IFN-+ T cells (a) and their expression gated on either total CD8+ cells or CD8+ IFN-+ cells (b) are shown. The numbers indicated in each quadrant are percentages. Experiments were performed on three individuals and representative results are shown.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Mapping of T-cell immunodominant peptides on the membrane protein of SARS-CoV by IFN- ELISpot assay in recovered SARS patients. PBMCs were isolated from fully recovered SARS patients and cultured in vitro with individual peptides (1 µg ml–1) from SARS-CoV M protein for IFN- ELISpot assay in the presence of anti-CD28 (1 µg ml–1) and anti-CD49d mAbs (1 µg ml–1). Results shown are representative of two out of four independent experiments. SFC, spot-forming cells.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Intracellular IFN- staining to confirm CD8+ T-cell immunodominant peptides of SARS-CoV M protein in recovered SARS patients. PBMCs were isolated from fully recovered SARS patients and cultured for 5 h with optimal individual peptides (1 µg ml–1). The immune response was evaluated by intracellular IFN- staining with anti-CD4, CD8 and IFN- antibodies. Numbers indicated in the upper right corner of each quadrant are percentages. Data shown are representative of three independent experiments with similar results.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

It is well known that CD4⁺ T cells can be divided into two major subpopulations, Th1 and Th2, based on cytokine production and biological functions (Abbas et al., 1996). Th1 cells are heterogeneous IFN-⁺ and IFN-^– cells. The IFN-^– Th1 cells may differentiate into memory cells in vivo (Wu et al., 2002). Consistent with the observations in an animal model (Jin et al., 2005), we found that 30 overlapping SARS-CoV M peptides induced a strong Th1 type response, reflected by expressing high levels of IFN- and IL-2. Based on the production of IFN- and IL-2, SARS-CoV M-specific CD4⁺ T cells could be divided into three distinct populations: single IFN--secreting cells, double IFN-/IL-2-secreting cells and single IL-2-secreting cells. IL-2 may play an important role in sustaining memory T cells and IFN- may mediate an effector function. Thus, IL-2⁺ T cells may be able to differentiate into long-term memory T cells, whereas the single IFN-⁺ T cells may be the effector and short-lived memory T cells. In agreement with other observations (Mallard et al., 2004; Zimmerli et al., 2005), SARS-CoV M-specific CD8⁺ T cells are not the major producer of IL-2. In this study, the majority of IFN-⁺ CD8⁺ T cells were CD45RO^– CCR7^–. This is supported by evidence that virus-specific IFN-/IL-2-secreting CD8⁺ T cells were CD45RA^– CCR7⁺,whereas single IFN--secreting CD8⁺ T cells were either CD45RA^– CCR7^– or CD45RA⁺ CCR7^– (Zimmerli et al., 2005).

It has been generally accepted that CD45RO expression defines activated or memory T cells. Here we found that SARS-CoV M-specific memory CD4⁺ T cells were mostly CD45RO⁺, while the majority of memory CD8⁺ T cells displayed CD45RO^– phenotype. These findings suggest that, at least in humans, the capacity of memory CD8⁺ T cells to express CD45RO in vivo may be different from that of CD4⁺ T cells. Wills et al. (1999) showed that virus-specific memory CD8⁺ T cells revert from CD45RO^high to CD45RA^high in vivo. Since CD45RA^high cells contain both naive cells and memory cells, we conclude that CD45RA is not a reliable marker of naive CD8⁺ T cells in human and that CD45RO is a marker of cell activation that does not identify all CD8⁺ memory T cells.

Based on the expression of cell-homing receptors, memory T cells can be divided into two distinct subsets: central memory T cells (T_CM) expressing CCR7 and CD62L, which allow efficient homing to lymph nodes, and effector memory T cells (T_EM), which lack the expression of these two lymph node homing receptors. In our study, the majority of IFN--producing CD4⁺ T cells were CCR7⁺. These cells with CD45RO⁺ CCR7⁺ phenotype are typical of T_CM and the producer of IL-2 (Wherry et al., 2003). Thus, we speculate that double IFN-/IL-2-secreting CD4⁺ T cells are T_CM expressing CD45RO⁺ CCR7⁺. In contrast, SARS-CoV M-specific CD8⁺ T cells predominantly displayed CD45RO^– and CCR7^– phenotype, which might represent terminally differentiated effector memory T cells (Champagne et al., 2001). This may reflect differences in the inherent stability of CD4⁺ and CD8⁺ memory T cells that are CCR7^– in normal steady-state conditions (Seder & Ahmed, 2003). The SSp-1 (an HLA-A*0201-restricted CD8⁺ T-cell epitope on SARS-CoV S protein)-specific CTL cells exhibited a differentiated effector phenotype, CD45RA⁺ CCR7^– CD62L^– (Chen et al., 2005). Consistent with those observations, we found that the vast majority of SARS-CoV M-specific memory CD8⁺ T cells were mostly confined to the CD45RO^– CCR7^– CD62L^– population. However, SARS-CoV M-specific memory CD4⁺ T cells expressed a mixed CCR7⁺ CD62L^– phenotype that differs from classical CCR7⁺ CD62L⁺ T_CM and CCR7^– CD62L^– T_EM cells. These studies suggest that the expression of CCR7 and CD62L in memory CD4⁺ T cells may overlap only partially.

In further study, we identified four human T-cell immunodominant peptides on the M protein of SARS-CoV, p21-44, p65-91, p117-140 and p200-220, which were able to induce cellular immunity mediated by CD4⁺ and CD8⁺ T cells. These responses were predominantly of memory CD8⁺ T cells, compared with memory CD4⁺ T cells. Together with the identification of the T-cell immunodominant peptides of the M protein of SARS-CoV, this may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for SARS.

In conclusion, SARS-CoV infection elicited T-cell responses to the M protein in recovered SARS patients, and this immunity persisted for a long period of time. Both CD4⁺ and CD8⁺ T cells were involved in the maintenance of immune memory to SARS-CoV M protein, which may have important implications in developing SARS vaccines.

	ACKNOWLEDGEMENTS

 
This work was supported by grants from the National Key Basic Research Program of China (2001CB510007), National Natural Science Foundation of China (No. 30340012), Guangdong Province of Science and Technology (No. 2003Z3—E0491), and Ministry of Education, Guangdong Province of China.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Abbas, A. K., Murphy, K. M. & Sher, A. (1996). Functional diversity of helper T lymphocytes. Nature 383, 787–793.[CrossRef][Medline]

Bisht, H., Roberts, A., Vogel, L., Bukreyev, A., Collins, P. L., Murphy, B. R., Subbarao, K. & Moss, B. (2004). Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proc Natl Acad Sci U S A 101, 6641–6646.[Abstract/Free Full Text]

Bukreyev, A., Lamirande, E. W., Buchholz, U. J., Vogel, L. N., Vogel, L. N., Elkins, W. R., StClaire, M., Murphy, B. R., Subbarao, K. & Collins, P. L. (2004). Mucosal immunization of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet 363, 2122–2127.[CrossRef][Medline]

Champagne, P., Ogg, G. S., King, A. S., Knabenhans, C., Ellefsen, K., Nobile, M., Appay, V., Rizzardi, G. P., Fleury, S. & other authors (2001). Skewed maturation of memory HIV-specific CD8 T lymphocytes. Nature 410, 106–111.[CrossRef][Medline]

Chen, H., Hou, J., Jiang, X., Ma, S., Meng, M., Wang, B., Zhang, M., Zhang, M., Tang, X. & other authors (2005). Response of memory CD8⁺ T cells to severe acute respiratory syndrome (SARS) coronavirus in recovered SARS patients and healthy individuals. J Immunol 175, 591–598.[Abstract/Free Full Text]

Drosten, C., Gunther, S., Preiser, W., van der Werf, S., Brodt, H. R., Becker, S., Rabenau, H., Panning, M., Kolesnikova, L. & other authors (2003). Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348, 1967–1976.[Abstract/Free Full Text]

Gao, W., Tamin, A., Soloff, A., D'Aluto, L., Nwanegbo, E., Robbins, P. D., Bellini, W. J., Barratt-Boyes, S. & Gambotto, A. (2003). Effects of a SARS-associated coronavirus vaccine in monkeys. Lancet 362, 1895–1896.[CrossRef][Medline]

He, Y., Zhou, Y., Siddiqui, P., Niu, J. & Jiang, S. (2005). Identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus. J Clin Microbiol 43, 3718–3726.[Abstract/Free Full Text]

Jin, H., Xiao, C., Chen, Z., Chen, Z., Kang, Y., Ma, Y., Zhu, K., Xie, Q., Tu, Y. & other authors (2005). Induction of Th1 type response by DNA vaccinations with N, M, and E genes against SARS-CoV in mice. Biochem Biophys Res Commun 328, 979–986.[CrossRef][Medline]

Ksiazek, T. G., Erdman, D., Goldsmith, C. S., Zaki, S. R., Peret, T., Emery, S., Tong, S., Urbani, C., Comer, J. A. & other authors (2003). A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348, 1953–1966.[Abstract/Free Full Text]

Li, G., Chen, X. & Xu, A. (2003). Profile of specific antibodies to the SARS-associated corona virus. N Engl J Med 349, 508–509.[Free Full Text]

Liu, X., Shi, Y., Li, P., Li, L., Yi, Y., Ma, Q. & Cao, C. (2004). Profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (SARS)-associated coronavirus in probable SARS patients. Clin Diagn Lab Immunol 11, 227–228.[CrossRef][Medline]

Mallard, E., Vernel-Pauillac, F., Velu, T., Lehmann, F., Abastado, J. P., Salcedo, M. & Bercovici, N. (2004). IL-2 production by virus- and tumor-specific human CD8 T cells is determined by their fine specificity. J Immunol 172, 3963–3970.[Abstract/Free Full Text]

Peng, H., Yang, L. T., Li, J., Lu, Z. Q., Wang, L. Y., Koup, R. A., Bailer, R. T. & Wu, C. Y. (2006a). Human memory T cell responses to SARS-CoV E protein. Microbes Infect 8, 2424–2431.[CrossRef][Medline]

Peng, H., Yang, L. T., Wang, L. Y., Li, J., Huang, J., Lu, Z. Q., Koup, R. A., Bailer, R. T. & Wu, C. Y. (2006b). Long-lived memory T lymphocyte responses against SARS coronavirus nucleocapsid protein in SARS-recovered patients. Virology 351, 466–475.[CrossRef][Medline]

Seder, R. A. & Ahmed, R. (2003). Similarities and differences in CD4⁺ and CD8⁺ effector and memory T cell generation. Nat Immunol 4, 835–842.[CrossRef][Medline]

Soo, Y. O., Cheng, Y., Wong, R., Hui, D. S., Lee, C. K., Tsang, K. K., Ng, M. H., Chan, P., Cheng, G. & Sung, J. J. (2004). Retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in SARS patients. Clin Microbiol Infect 10, 676–678.[CrossRef][Medline]

Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W. J., Zaki, S. & Murphy, B. (2004). Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice. J Virol 78, 3572–3577.[Abstract/Free Full Text]

Takasuka, N., Fujii, H., Takahashi, Y., Kasai, M., Morikawa, S., Itamura, S., Ishii, K., Sakaguchi, M., Ohnishi, K. & other authors (2004). A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice. Int Immunol 16, 1423–1430.[Abstract/Free Full Text]

Wang, B., Chen, H. B., Jiang, X. D., Zhang, M., Wan, T., Li, N., Zhou, X., Wu, Y., Yang, F. & other authors (2004a). Identification of an HLA-A*0201-restricted CD8⁺ T-cell epitope SSp-1 of SARS-CoV spike protein. Blood 104, 200–206.[Abstract/Free Full Text]

Wang, Y. D., Sin, W. Y., Xu, G. B., Yu, J., Meng, L., Yang, R. F., Lai, S. T., Guo, Z. H., Xie, Y. & other authors (2004b). T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus spike protein elicit a specific T-cell immune response in patients who recovered from SARS. J Virol 78, 5612–5618.[Abstract/Free Full Text]

Wang, Z., Yuan, Z. H., Matsumoto, M., Hengge, U. R. & Chang, Y. F. (2005). Immune responses with DNA vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in BALB/c mice. Biochem Biophys Res Commun 327, 130–135.[CrossRef][Medline]

Wherry, E. J., Teichgräber, V., Becker, T. C., Masopust, D., Kaech, S. M., Antia, R., von Andrian, U. H. & Ahmed, R. (2003). Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol 4, 225–234.[CrossRef][Medline]

Wills, M. R., Carmichael, A. J., Weekes, M. P., Mynard, K., Okecha, G., Hicks, R. & Siaaona, J. G. (1999). Human virus-specific CD8⁺ CTL clones revert from CD45RO^high to CD45RA^high in vivo: CD45RA^high CD8⁺ T cells comprise both naïve and memory cells. J Immunol 162, 7080–7087.[Abstract/Free Full Text]

Wu, C. Y., Kirman, J. R., Rotte, M. J., Davey, D. F., Perfetto, S. P., Rhee, E. G., Freidag, B. L., Hill, B. J., Douek, D. C. & Seder, R. A. (2002). Distinct lineages of T_H1 cells have differential capacities for memory cell generation in vivo. Nat Immunol 3, 852–858.[CrossRef][Medline]

Yang, Z. Y., Kong, W. P., Huang, Y., Roberts, A., Murphy, B. R., Subbarao, K. & Nabel, G. J. (2004). A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature 428, 561–564.[CrossRef][Medline]

Yang, L. T., Peng, H., Zhu, Z. L., Li, G., Huang, Z. T., Zhao, Z. X., Koup, R. A., Bailer, R. T. & Wu, C. Y. (2006). Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients. Clin Immunol 120, 171–178.[CrossRef][Medline]

Zhi, Y., Kobinger, G. P., Jordan, H., Suchma, K., Weiss, S. R., Shen, H., Schumer, G., Gao, G., Boyer, J. L. & other authors (2005). Identification of murine CD8 T cell epitopes in codon-optimized SARS-associated coronavirus spike protein. Virology 335, 34–45.[CrossRef][Medline]

Zimmerli, S. C., Harari, A., Cellerai, C., Vallelian, F., Bart, P. A. & Pantaleo, G. (2005). HIV-1-specific IFN-/IL-2-secreting CD8 T cells support CD4-independent proliferation of HIV-1-specific CD8 T cells. Proc Natl Acad Sci U S A 102, 7239–7244.[Abstract/Free Full Text]

Received 9 January 2007; accepted 1 June 2007.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Yang, L. Articles by Wu, C. Search for Related Content PubMed PubMed Citation Articles by Yang, L. Articles by Wu, C. Agricola Articles by Yang, L. Articles by Wu, C.