Development of Zika viral pseudoinfectious virus as Zika vaccine candidate

寨卡病毒假感染性病毒作为寨卡疫苗候选物的开发 摘要 寨卡病毒（ZIKV）是一种蚊媒黄病毒，于1947年在乌干达首次发现。然后它传播开来 亚洲和太平洋。近年来，美洲爆发的疫情及其对吉兰·巴雷的影响 (GBS)综合征和母亲感染后出生的小头症婴儿导致世卫组织 宣布ZIKV感染是“国际关注的突发公共卫生事件”。目前还 没有具体的治疗。因此，开发安全有效的ZIKV感染疫苗是当务之急。 公共卫生的。在此，我们提出基于ZIKV开发新型ZIKV疫苗候选物。 通过将ZIKV亚基因组复制子掺入病毒颗粒中产生的假感染性病毒（PIV）， 稳定的包装细胞。该方法基于我们在登革热病毒（DENV）PIV方面的丰富经验 调查在DENV PIV生产系统中，通过缺失DENV PIV的亚基因组复制子来构建亚基因组复制子。 衣壳蛋白（C）基因，并优化前衣壳蛋白（C）的信号肽序列。 膜蛋白（prM），以促进病毒颗粒的形成。包装细胞被开发用于 双蛋白Cpr的诱导型表达，其中蛋白pr是病毒蛋白prM的“pr”区段 将蛋白质C固定在内质网上。当复制子被引入到 在包装细胞中，通过复制子编码的病毒蛋白酶从双蛋白Cpr释放蛋白C。 复制子和包装细胞协调表达病毒结构蛋白， 将复制子掺入病毒颗粒中以产生DENV PIV。为了开发ZIKV PIV，我们 已经通过化学合成构建了ZIKV的全长cDNA克隆。基于这些高度 有希望的结果，这个第一阶段STTR建议的具体目标是：1）构建亚基因组 2）基于FDA批准的Vero细胞的稳定包装细胞系的开发，3）快速和 从感染的包装线有效收获ZIKV PIV，以及4）安全性的初步分析 和免疫原性。成功完成该计划将 启动第二阶段研究，包括开发用于PIV生产的具有成本效益的GMP工艺， 工业规模和对ZIKV感染的免疫力的临床前动物研究。

Development of Zika viral pseudoinfectious virus as zika vaccine candidate Abstract Zika virus(ZIKV) is a mosquito-borne flavivirus that was first identified in Uganda in 1947. It then spread to Asia and Pacific. Recently, the explosive outbreaks in Americas and its implication in Guillan Barré (GBS) syndrome and the birth of microcephalic infants following maternal infection lead to WHO declared that ZIKV infection is “a Public Health Emergency of International Concern“. Currently, there is no specific treatment. Thus, developing a safe and effective vaccine for ZIKV infection is a high priority for public health. Here, we propose to develop a novel ZIKV vaccine candidate based on ZIKV pseudoinfectious virus (PIV) produced by incorporation of ZIKV subgenomic replicon into viral particle in stable packaging cells. The approach is based on our extensive experience of dengue virus (DENV) PIV investigation. In DENV PIV production system, the subgenomic replicon was constructed by deleting the capsid protein (C) gene from the DENV genome and optimizing the signal peptide sequence of pre- membrane protein (prM) to facilitate the formation of viral particles. Packaging cells were developed for inducible expression of a bi-protein Cpr, where the protein pr is the “pr” segment of viral protein prM that holds the protein C on the endoplasmic reticulum (ER). When the replicon was introduced into the packaging cells, protein C was released from the bi-protein Cpr by a replicon-encoded viral protease. Coordinate expression of viral structural proteins by the replicon and packaging cells led to the incorporation of the replicon into viral particle to produce DENV PIVs. Toward to develop a ZIKV PIV, we have constructed a full-length cDNA clone of ZIKV by chemical synthesis. Based upon these highly promising results, the Specific Aims of this Phase I STTR proposal are: 1) construction of subgenome for ZIKV 2) development of stable packaging cell lines based on FDA approved Vero cells, 3) rapid and efficient harvesting of ZIKV PIV from infected packaging lines, and 4) preliminary analysis of the safety and immunogenicity of the proposed vaccine in mice. Successful completion of this program will enable a Phase II research including development of a cost-effective GMP process for PIVs production at industrial scale and a pre-clinical animal study of immunity to ZIKV infection.