Identifying epitopes responsible for neutralizing antibody and DC-SIGN binding on the spike glycoprotein of the severe acute respiratory syndrome coronavirus
Identifying epitopes responsible for neutralizing antibody and DC-SIGN binding on the spike glycoprotein of the severe acute respiratory syndrome coronavirus
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DOI：
10.1128/jvi.01138-06
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发表时间：
2006-11-01
影响因子：
5.4
通讯作者：
Chen, Yi-Ming Arthur
中科院分区：
文献类型：
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作者：
Shih, Yi-Ping;Chen, Chia-Yen;Chen, Yi-Ming Arthur
严重急性呼吸综合征相关冠状病毒（SARS-CoV）使用树突状细胞特异性ICAM-3抓取非整合素（DC-SIGN）通过细胞受体血管紧张素转换酶2促进细胞进入。在这个项目中，我们使用了表达不同长度SARS-CoV刺突蛋白的重组杆状病毒，在捕获试验中推导出最小的DC-SIGN结合区域。我们的结果确定了S蛋白的氨基酸（aa）残基324至386之间的区域位置。然后，我们产生了9个单克隆抗体（MAb）对S蛋白定位DC-SIGN结合域使用捕获试验与假型病毒，并观察到MAb SIa 5显着阻断S蛋白-DC-SIGN相互作用。使用HKU 39849 SARS-CoV株和人未成熟树突状细胞的增强试验证实了我们的观察。来自pepscan分析和M13噬菌体肽展示文库系统的数据将反应性MAb SIa 5表位定位于S蛋白的aa残基363至368。来自测试具有突变的S蛋白的N-连接糖基化位点的三种假型病毒的捕获测定的结果表明，仅两种假型病毒（N330 Q和N357 Q，两者都在SIa 5表位附近丢失糖基化位点）具有降低的DC-SIGN结合能力。我们还注意到，单克隆抗体SIb 4对HKU 39849产生中和作用;其反应性表位定位于S蛋白的氨基酸残基435至439。我们提供的数据，以促进治疗剂和预防性疫苗对SARS冠状病毒感染的发展。
The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) uses dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) to facilitate cell entry via cellular receptor-angiotensin-converting enzyme 2. For this project, we used recombinant baculoviruses expressing different lengths of SARS-CoV spike (S) protein in a capture assay to deduce the minimal DC-SIGN binding region. Our results identified the region location between amino acid (aa) residues 324 to 386 of the S protein. We then generated nine monoclonal antibodies (MAbs) against the S protein to map the DC-SIGN-binding domain using capture assays with pseudotyped viruses and observed that MAb SIa5 significantly blocked S protein-DC-SIGN interaction. An enhancement assay using the HKU39849 SARS-CoV strain and human immature dendritic cells confirmed our observation. Data from a pepscan analysis and M13 phage peptide display library system mapped the reactive MAb SIa5 epitope to aa residues 363 to 368 of the S protein. Results from a capture assay testing three pseudotyped viruses with mutated N-linked glycosylation sites of the S protein indicate that only two pseudotyped viruses (N330Q and N357Q, both of which lost glycosylation sites near the SIa5 epitope) had diminished DC-SIGN-binding capacity. We also noted that MAb SIb4 exerted a neutralizing effect against HKU39849; its reactive epitope was mapped to aa residues 435 to 439 of the S protein. We offer the data to facilitate the development of therapeutic agents and preventive vaccines against SARS-CoV infection.