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。它在亚洲广泛传播，并正在超越其传统边界。目前还没有针对乙脑的特异性治疗方法。目前，有3种乙脑疫苗在不同国家使用，但国际上只有一种是小鼠脑源性灭活疫苗。虽然这种疫苗在减少乙脑发病率方面有效，但它相对昂贵，并与严重的过敏和神经反应有关。出于监管方面的考虑，另外两种乙脑疫苗仅在中国使用。乙脑疫苗开发是世界卫生组织（WHO）工作重点之一。迄今为止，还没有研究使用不能复制的乙脑病毒粒子作为免疫原来刺激有效免疫。我们最近开发了一种高效的DNE2/AC复制子封装细胞系。假传染性病毒样颗粒（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，也完全没有任何传染性病毒。因此，为了开发更安全、更有效、成本更低的乙脑疫苗，我们构建了乙脑病毒（JEV） SA14-14-2株全长感染性cDNA克隆。基于这些非常有希望的结果，本一期SBIR计划的具体目标是：1)构建JEV SA14-14-2株的JEV/AC复制子；2)培养提供乙脑结构蛋白C的稳定包装细胞系；3)从感染的包装细胞系中收获乙脑病毒PVLP，分析包装细胞系和PVLP传代过程中的稳定性；4)小鼠免疫原性初步分析。该项目的成功完成将使SBIR II期研究成为可能，包括开发具有成本效益的工业规模PVLP生产GMP工艺，体内决定因子作为免疫原的滴定，评估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。

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.