Development of peptide-based vaccine against SARS-CoV

描述(由申请人提供)： 由冠状病毒引起的严重急性呼吸综合征(SARS)是一种在全球范围内流行的人类新发传染病。因此，迫切需要实际的诊断方法、预防和治疗疫苗以及抗病毒药物。本研究的目的是开发基于多肽的疫苗，包括重叠多肽库的组合合成、B细胞和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，根据BJ01蛋白序列合成了1938个重叠的单肽(长度为10个氨基酸)，包括刺突糖蛋白、膜蛋白、囊膜蛋白和核衣壳蛋白，它们可能构成SARS冠状病毒的人类免疫识别结构域。 2.采用ELISA法直接测定B细胞表位。与至少12份抗体阳性的SARS患者血清反应的多肽将被确定为阳性。 3.通过多肽刺激SARS康复者外周血单个核细胞(PBMC)释放细胞因子，用ELISPOT法检测干扰素-γ、白介素2、白介素12等，以确定上述蛋白的SARS冠状病毒特异性T细胞表位。 4.将设计、合成覆盖两个或三个已识别表位的拉长多肽，并测试其抗原性。当动物血清中的抗肽(肽-载体结合物)抗体中和SARS冠状病毒时，将确定它们的免疫原性。 5.将同时含有B细胞中和抗原和SARS冠状病毒特异性T细胞表位的多种抗原共价偶联到蛋白载体上，从而实现对动物的免疫。将对SARS冠状病毒攻击的保护性和CTL反应进行调查，以评估基于多肽的疫苗。

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.