Development of peptide-based vaccine against SARS-CoV

描述（由申请人提供）： 严重急性呼吸综合征（severe acute respiratory syndrome，SARS）是由冠状病毒引起的一种在全球范围内迅速蔓延的人类传染病。因此，迫切需要实用的诊断方法、预防和治疗性疫苗以及抗病毒药物。本研究的主要目的是开发肽疫苗，包括重叠肽库的组合合成、B和T细胞表位的直接鉴定、中和抗原的筛选和动物免疫。 1.利用我们的编码和“MBGB”组合技术，将根据BJ 01蛋白序列合成总共1938个重叠的单个肽（长度为10个氨基酸），包括刺突糖蛋白、膜蛋白、包膜蛋白和核衣壳蛋白，其可能包含SARS CoV的人免疫识别结构域。 2.将使用ELISA方法直接测定B细胞表位。与至少12个SARS患者抗体阳性血清反应的肽将被确定为阳性。 3.通过ELISA方法检测SARS康复患者外周血单个核细胞（PBMC）释放细胞因子（如IFN-γ、IL-2或IL-12）的肽的刺激作用，鉴定来自上述蛋白的SARS CoV特异性T细胞表位。 4.将设计、合成覆盖两个或三个鉴定的表位的延长肽并测试其抗原性。当来自动物血清的抗肽（肽-载体缀合物）抗体中和SARS CoV时，将确定它们的免疫原性。 5.含有B细胞中和抗原和SARS CoV特异性T细胞表位的多个抗原将共价结合到能够免疫动物的蛋白载体上。将研究SARS CoV攻击和CTL应答的保护以评估基于肽的疫苗。

DESCRIPTION (provided by applicant): Severe acute respiratory syndrome (SARS) caused by coronavirus is an emerging human infectious disease that epidemically spread worldwide. Therefore, the actual diagnostic method, preventing and therapeutic vaccine, and antiviral drugs are urgently demanded. This proposal aims at development of peptide-based vaccine including combinatorial synthesis of overlapped peptide library, identification of B- and T-cell epitopes directly, selection of neutralizing antigens and immunization of animal. 1.Using our coding and "MBGB" combinatorial technology, total 1938 overlapped individual peptides (10 amino acids in length) will be synthesized according to the BJ01 protein sequence including spike glycoprotein, membrane protein, envelope protein, and nucleocapasid protein, which potentially comprise the human immuno-recognizing domains of SARS CoV. 2. B-cell epitopes will be directly determined using ELISA method. The peptides reacted across with at least twelve antibody-positive sera of SARS patients will be determined as being positive. 3. SARS CoV specific T-cell epitopes from above proteins will be identified by peptide's stimulation of peripheral blood mononuclear cells (PBMC) releasing cytokines of recovered SARS patients, such as IFN-gamma, IL2, or IL12 detecting by Elispot method. 4. The elongated peptides covering two or three identified epitopes will be designed, synthesized and tested their antigenicity. Their immunogenicity will be determined when the anti-peptide (peptide-carrier conjugate) antibody from animal sera neutralizes the SARS CoV. 5. Multiple antigens containing both B-cell neutralizing antigens and SARS CoV specific T-cell epitopes will covalently conjugate onto protein carrier which will be able to immunize animals. The protection of SARS CoV challenges and CTL responses will be investigated to evaluate the peptide-based vaccine.