Development of Pseudoinfectious VLP as Novel JEV Vaccine

描述（由申请人提供）：日本脑炎（JE）是世界上最重要的病毒性脑炎。它在整个亚洲广泛传播，并且正在超越其传统边界。日本脑炎没有特效治疗方法。目前，不同国家使用的乙脑疫苗有3种，但国际上只有一种，即鼠脑灭活疫苗。虽然这种疫苗可以有效降低乙脑的发病率，但它相对昂贵，并且与严重的过敏和神经反应有关。出于监管方面的考虑，其他 2 种乙脑疫苗仅在中国使用。乙脑疫苗的开发被世界卫生组织（WHO）列为高度优先事项。迄今为止，还没有研究采用无法复制的乙脑病毒病毒粒子作为免疫原来刺激有效的免疫力。我们最近开发了一种高效的 DNE2/AC 复制子包装细胞系。假感染性病毒样颗粒 (PVLP) 在 BHK-21 细胞上的传代表明，即使在包装细胞上传代 6 次后，也完全不存在任何感染性病毒。因此，为了实现开发更安全、更有效、成本更低的乙脑疫苗的总体目标，我们构建了乙脑病毒（JEV）SA14-14-2株的全长感染性cDNA克隆。基于这些极具前景的结果，第一阶段 SBIR 提案的具体目标是： 1) 构建 JEV SA14-14-2 株的 JEV/AC 复制子； 2) 开发在列车中提供JE结构蛋白C的稳定包装细胞系； 3)从感染的包装细胞系中收获JEV PVLP，并分析包装细胞系和PVLP在传代过程中的稳定性； 4) 初步分析所提出的疫苗在小鼠中的免疫原性。该计划的成功完成将使SBIR II期研究成为可能，包括开发用于工业规模PVLP生产的具有成本效益的GMP工艺、作为体内免疫原的决定簇的滴定、评估对PVLP的体液和细胞反应，以及对乙脑感染免疫的临床前动物研究。 伪传染性病毒样颗粒作为候选疫苗本身就是安全性和有效性的最佳组合。低成本生产对于实际应用至关重要。在本提案中，我们将开发一种可用于大规模生产的高效包装系统。

DESCRIPTION (provided by applicant): Japanese encephalitis (JE) is the most important viral encephalitis in the world. It is widespread throughout Asia and is spreading beyond its traditional boundaries. There is no specific treatment for JE. Currently, 3 kinds of JE vaccine are in use in different countries, but only one is available internationally, a mouse-brain-derived inactivated vaccine. Although, this vaccine has been effective in reducing the incidence of JE, it is relatively expensive and has been linked to severe allergic and neurological reactions. Other 2 JE vaccines are only used in China, due to regulatory concern. Vaccine development for JE is a high priority on the list of World Health Organization (WHO). To date, no study has employed a replication-incompetent JEV virion as immunogens to stimulate effective immunity. We have recently developed a highly efficient packaging cell line for DNE2/AC replicon. Passaging of the pseudoinfectious virus-like particles (PVLP) on BHK-21 cell illustrated the complete absence of any infectious virus, even after 6 passages on packaging cells. Thus, toward the overall goal of developing a safer, more effective, and less costly JE vaccine, we have constructed a full-length infectious cDNA clone for JE virus (JEV) SA14-14-2 strain. Based upon these highly promising results, the Specific Aims of this Phase I SBIR proposal are: 1) construction of JEV/AC replicon for JEV SA14-14-2 strain; 2) development of stable packaging cell lines providing JE structural protein C in trains; 3) harvest of JEV PVLPs from infected packaging cell line, and analyzing the stability of the packaging cell lines and PVLP during passages; 4) preliminary analysis of the immunogenicity of the proposed vaccine in mice. Successful completion of this program will enable SBIR Phase II research including development of a cost-effective GMP process for PVLP production at industrial scale, titration of the determinants as immunogens in vivo, assessment of both the humoral and cellular responses to the PVLP, and a pre-clinical animal study of immunity to JE infection. Pseudoinfectious virus-like particle as vaccine candidate is inherent optimal combination of safety and efficacy. Low cost production is critical for practical use. In this proposal, we are going to develop a high efficient packaging system that can be used for large scale production.