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

NS3含有与胰蛋白酶样丝氨酸序列同源的四个区域 蛋白酶NS3中这四个区域中的两个（“框”3和4）包含 预测对底物结合重要的残基（AA 1604，1605， 1625，1627和1628）。 我们诱变了DEN2中的一些残基， 表征它们对蛋白酶活性的需求，并鉴定 具有部分切割缺陷的突变体。 总共有46个突变， 分析了 框4中的突变通常消除蛋白酶活性， 这与框4构成了 基质结合口袋。 相反，框3中的许多突变， 包括AA 1604和1605中的那些，保留了显著的蛋白酶活性。 在框3中的其他突变，聚集在催化丝氨酸周围， 乳沟 这证实了框3中的一些保留对于 蛋白酶功能，但与aa 1604的拟议作用不一致 和1605在衬底结合。目前正在开展工作， 具有部分活性的突变回到DEN2，以确认 所观察到的对蛋白酶功能的影响以及可能减弱 病毒 LVVD的主要监管职责包括 评价用于开发活病毒疫苗的IND， 对抗由黄病毒引起的疾病（例如，登革热病毒，蜱传 脑炎病毒，日本脑炎病毒），甲病毒（例如， 基孔肯雅病毒、东部和委内瑞拉马脑炎病毒）， 和沙粒病毒（例如，朱宁病毒，汉坦病毒）。 这些都是 正链RNA病毒。 因此，一个可行的战略， 针对任何这些病毒的疫苗是为了确定潜在的减毒 突变，并使用感染性cDNA将它们改造成病毒基因组， 分身 此外，适用于识别和 假定疫苗株的检测是相同的，无论 技术来获取它们。 因此，本文中描述的工作 该项目将是非常宝贵的，帮助我们批判的方法， 另一些人则用来衍生疫苗病毒并对其进行评估。 另外我们 寻求开发我们自己的减毒登革热病毒， 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.