DENGUE VACCINE DEVELOPMENT--MUTATIONS IN NONSTRUCTURAL PROTEIN REGION OF DEN2

NS3包含四个与类胰酶丝氨酸序列同源的区域 蛋白酶。NS3中这四个区域中的两个(方框3和4)包含 预测对底物结合很重要的残基(AA 1604,1605， 1625、1627和1628)。我们将DEN2中的一些残基突变为 表征他们对蛋白水解酶活性的需求并确定 有部分切割缺陷的突变体。总共有46个突变是 分析过了。框4中的突变通常会使蛋白酶活性丧失， 与框4构成部分的假设一致 衬底捆绑袋。相比之下，框3中的许多突变， 包括AA 1604和1605中的那些，都保持了显著的蛋白酶活性。 框3中的其他突变，聚集在催化丝氨酸周围，废除 乳沟。这证实了框3中的某些阻力对于 蛋白水解酶功能，但与AA 1604的建议作用不一致 底物结合时为1605。目前正在开展工作，推出一些 带有部分活性的突变返回DEN2，以努力确认 观察到的对蛋白酶功能的影响，可能是为了减弱 病毒。 LVVD的主要监管责任包括 INDS用于开发保护性活病毒疫苗的评价 预防由黄病毒引起的疾病(例如，壁虱传播的登革热病毒) 脑炎病毒、日本脑炎病毒)、甲型病毒(例如， 基孔肯雅病毒、东部和委内瑞拉马脑炎病毒)、 和阿拉伯病毒(例如，Junin病毒、汉坦病毒)。这些都是 正链RNA病毒。因此，一个可行的战略是生产一个 针对这些病毒的疫苗是为了识别潜在的减毒作用 突变，并使用具有感染性的cDNA将它们工程到病毒基因组中 克隆人。此外，适用于识别和识别的原则 对假定的疫苗毒株的测试是相同的，无论 用来衍生它们的技术。因此，本文件中描述的工作 项目将在帮助我们批评 其他人则获得疫苗病毒并对其进行评估。此外，我们 寻求开发我们自己的减毒登革热病毒作为候选 疫苗研发。

NS3 contains four regions of sequence homology to trypsin-like serine proteases. Two of these four regions in NS3 ("boxes" 3 and 4) contain residues predicted to be important for substrate binding (aa 1604, 1605, 1625, 1627 and 1628). We mutagenized some of these residues in DEN2 to characterize their requirement for protease activity and to identify mutants with partial cleavage defects. In all, 46 mutations were analyzed. Mutations in box 4 generally abolish protease activity, consistent with the hypothesis that box 4 forms part of the substrate-binding pocket. In contrast, many of the mutations in box 3, including those in aa 1604 and 1605, retain significant protease activity. Other mutations in box 3, clustered around the catalytic serine, abolish cleavage. This confirms that some resisues in box 3 are important for protease function, but is inconsistent with the proposed role for aa 1604 and 1605 in substrate binding. Work is underway to introduce some of the mutations with partial activity back into DEN2, in an effort to confirm the observed effect on protease function and possibly to attenuate the virus. A major portion of the regulatory responsibilities of LVVD includes the evaluation of INDs for the development of live virus vaccines to protect against diseases caused by flaviviruses (e.g., dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus), alphaviruses (e.g., Chikungunya virus, Eastern and Venezuelan equine encephalitis viruses), and arenaviruses (e.g., Junin virus, Hantaviruses). These are all positive-stranded RNA viruses. Thus, a viable strategy for producing a vaccine against any of these viruses is to identify potential attenuating mutations and engineer them into the viral genome using an infectious cDNA clone. Moreover, the principles applied to the identification and testing of putative vaccine strains are identical, regardless of the technology employed to derive them. Thus, the work described in this project will be invaluable in helping us to critique methods used by others to derive vaccine viruses and to evaluate them. In addition, we seek to develop our own attenuated dengue viruses as candidates for vaccine development.