RECOMBINANT DENGUE VIRUS ANTIGEN PRODUCTION

登革热病毒（DEN）是一种引起登革热的黄病毒，登革热是一种急性发热性疾病，在全世界热带和亚热带地区发病率越来越高。4种DEN血清型和其他黄病毒通常在流行地区共传播，再加上它们广泛的抗原交叉反应性，混淆了血清诊断。由于以前暴露于不同的登革出血热血清型可能导致严重后果（登革出血热/DSS），因此确定登革出血热血清型对于指示患者治疗策略和预测登革出血热/DSS暴发非常重要。市售的诊断测试依赖于全病毒抗原，仅针对黄病毒。利用编码截断的DEN包膜蛋白与大肠杆菌MBP融合的质粒，开发针对每种DEN血清型的重组DEN抗原（rDEN）的商业生产方法。实验室规模的纯化工作将确定亲和层析条件，产生未聚集的重组蛋白；随后将扩大到30升的生产规模。抗原将通过Western blotting、dot EIA和酶联免疫吸附试验（ELISA）对人血清和小鼠抗体进行特异性和亲和力评估，这些抗体代表了由IFA、ELISA和中和试验确定的一系列特异性和滴度。将启动稳定性测试。将rDEN纳入我们的商业ELISA测试将提供所需的特异性，并避免费力和危险的病毒产生。拟议的商业应用：登革热是一种主要的公共卫生威胁，估计每年有超过1亿例病例，其地理范围正在扩大。广泛用作诊断辅助手段的血清学检测依赖于相对非特异性的全病毒抗原。建议用重组抗原替代这些抗原，这将通过贡献特异性显著提高登革热的检测。PanBio index已经在40多个国家分发登革热ELISA试剂盒，预计市场将扩大。改进后的酶联免疫吸附试验应得到广泛的接受。

Dengue virus (DEN) is a flavivirus causing dengue fever, an acute febrile illness which occurs with increasingly high incidence in the tropics and subtropics worldwide. The 4 DEN serotypes and other flaviviruses commonly cocirculate in endemic regions, which together with their extensive antigenic cross reactivity confound serodiagnosis. Because severe outcomes (DHF/DSS) may result from previous exposure to a different DEN serotype, DEN serotype identification is important in indicating patient treatment strategies and in predicting DHF/DSS outbreaks. Commercially available diagnostic tests rely on whole virus antigens which are only flavivirus-specific. Methods will be developed for commercial production of recombinant DEN antigens (rDEN) specific for each DEN serotype using plasmids encoding truncated DEN envelope proteins fused with E. coli MBP. Bench-scale purification efforts will define affinity chromatography conditions which yield unaggregated recombinant proteins; scale up to 30-liter production lots will follow. Antigens will be evaluated for specificity and avidity by Western blotting, dot EIA, and microplate ELISA with human sera and mouse antibodies representing a range of specificities and titers determined by IFA, ELISA, and neutralization tests. Stability tests will be initiated. Incorporation of rDEN into our commercial ELISA tests will provide needed specificity and avoid laborious and hazardous virus production. PROPOSED COMMERCIAL APPLICATIONS: Dengue is a major public health threat, with over 100 million cases estimated per year, and its geographic range is expanding. Serologic tests, widely used as diagnostic aids, rely on relatively nonspecific whole virus antigens. Replacement of these with recombinant antigens is proposed, this will significantly enhance detection of dengue by contributing specificity. PanBio InDx already distributes dengue ELISA kits in over 40 countries and the market is expected to expand. The improved ELISA tests should receive wide acceptability.