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Screening and Identification of Severe Acute Respiratory Syndrome-Associated Coronavirus-Specific CTL Epitopes¹

Minghai Zhou^*,¶, Dongping Xu, Xiaojuan Li^*, Hongtao Li^*,¶, Ming Shan^*, Jiaren Tang, Min Wang, Fu-Sheng Wang, Xiaodong Zhu^*,¶, Hua Tao^*, Wei He, Po Tien^2,* and George F. Gao^2,*,

^* Center for Molecular Immunology and Center for Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing, The People’s Republic of China; Beijing Institute of Infectious Diseases, Beijing 302 Hospital, Beijing, People’s Republic of China; Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, People’s Republic of China; Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford University, Oxford, United Kingdom; and ^¶ Graduate School, CAS, Beijing, People’s Republic of China

Severe acute respiratory syndrome (SARS) is a highly contagious and life-threatening disease that emerged in China in November 2002. A novel SARS-associated coronavirus was identified as its principal etiologic agent; however, the immunopathogenesis of SARS and the role of special CTLs in virus clearance are still largely uncharacterized. In this study, potential HLA-A*0201-restricted spike (S) and nucleocapsid protein-derived peptides were selected from an online database and screened for potential CTL epitopes by in vitro refolding and T2 cell-stabilization assays. The antigenicity of nine peptides which could refold with HLA-A*0201 molecules was assessed with an IFN- ELISPOT assay to determine the capacity to stimulate CTLs from PBMCs of HLA-A2⁺ SARS-recovered donors. A novel HLA-A*0201-restricted decameric epitope P15 (S411–420, KLPDDFMGCV) derived from the S protein was identified and found to localize within the angiotensin-converting enzyme 2 receptor-binding region of the S1 domain. P15 could significantly enhance the expression of HLA-A*0201 molecules on the T2 cell surface, stimulate IFN--producing CTLs from the PBMCs of former SARS patients, and induce specific CTLs from P15-immunized HLA-A2.1 transgenic mice in vivo. Furthermore, significant P15-specific CTLs were induced from HLA-A2.1-transgenic mice immunized by a DNA vaccine encoding the S protein; suggesting that P15 was a naturally processed epitope. Thus, P15 may be a novel SARS-associated coronavirus-specific CTL epitope and a potential target for characterization of virus control mechanisms and evaluation of candidate SARS vaccines.

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