Development of Zika viral pseudoinfectious virus as Zika vaccine candidate

寨卡病毒伪传染性病毒作为寨卡病毒候选疫苗的研究进展 摘要 寨卡病毒(ZIKV)是一种蚊媒黄病毒，1947年首次在乌干达发现。然后它传播开来 前往亚洲和太平洋地区。最近，美洲爆发的疫情及其对吉兰·巴雷的影响 (GBS)综合征和产妇感染后出生的小头症婴儿导致世卫组织 宣布寨卡病毒感染是“国际关注的突发公共卫生事件”。目前，有 没有特别的治疗方法。因此，开发安全有效的寨卡病毒感染疫苗是当务之急。 为了公众健康。在这里，我们建议开发一种基于ZIKV的新型ZIKV疫苗候选 ZIKV亚基因组复制子掺入病毒颗粒产生的伪感染病毒 稳定的包装细胞。该方法基于我们在登革病毒(DENV)PIV方面的丰富经验 调查。在DENV PIV生产系统中，亚基因组复制子是通过删除 新城疫病毒衣壳蛋白(C)基因克隆及前病毒信号肽序列的优化 膜蛋白(PRM)，促进病毒颗粒的形成。包装单元的开发是为了 双蛋白CPR的可诱导表达，其中Pr是病毒蛋白PrM的Pr片段 内质网(ER)上的蛋白C。当复制图标被引入到 在包装细胞中，蛋白C通过复制子编码的病毒蛋白酶从双蛋白CPR中释放出来。 复制子和包装细胞协调表达病毒结构蛋白导致 将复制子掺入病毒颗粒以产生DENV PIV。为了开发ZIKV PIV，我们 用化学合成法构建了ZIKV的全长cDNA克隆。基于这些高度的 结果令人振奋，这个第一阶段STTR提案的具体目标是：1)构建亚基因组 对于ZIKV 2)基于FDA批准的Vero细胞开发稳定的包装细胞系，3)快速和 从受感染的包装线上高效采集ZIKV PIV，以及4)安全性的初步分析 以及所提议的疫苗在小鼠中的免疫原性。成功完成这项计划将 支持第二阶段研究，包括为PIV生产开发具有成本效益的GMP流程 工业规模和对寨卡病毒感染免疫的临床前动物研究。

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.