Development of hantavirus vaccine and rapid diagnosis kit by using soluble recombinant envelope glycoproteins

1. 利用CAG启动子表达系统，证实重组汉坦病毒包膜糖蛋白G1和G2 （GPs）具有细胞融合活性。发现gp上的融合负责表位位于中和（FRNT）相关表位。因此，重组GPs被认为具有中和和融合相关的表位。此外，利用相同的载体，汉坦病毒核衣壳蛋白(N)也在哺乳动物细胞中表达。因此，N蛋白可能抑制包膜蛋白的表达和运输。结果表明，病毒样颗粒不是由gp和N蛋白单独形成的。利用重组全球定位蛋白（gp），制备了被变异为VSV G蛋白的汉坦病毒全球定位蛋白包膜的伪水疱性口炎病毒（VSV） （VSVΔG*HTN）。用重组汉城病毒（VSVΔG*SEO）的GPs制备了相似的伪VSV。这些假病毒作为真病毒的安全替代品应用于快速中和试验。这些伪病毒颗粒也用于疫苗接种，作为真病毒粒子的替代品。用可溶性重组GPs或假病毒粒子免疫小鼠。用可溶性重组GPs免疫小鼠，未检测到FRNT抗体。相反，VSVΔG*HTN病毒粒子免疫小鼠，检测到FRNT抗体。采用sc法接种4株汉坦病毒，对汉坦病毒进行攻毒。这些小鼠未表现出抗n抗体和汉坦病毒特异性CD8 T细胞反应的升高，表明FRNT抗体可以保护小鼠免受汉坦病毒感染。另一方面，所有接种VSVΔG*G或PBS的对照小鼠均无法逃脱汉坦病毒感染。结果表明，在VSV颗粒上包装的重组GPs可以诱导保护性FRNT抗体。本研究为研制难以制备真病毒颗粒的病毒疫苗提供了新的途径。

1. It was clarified that recombinant Hantavirus envelope glycoproteins G1 and G2 (GPs) had the cell-fusion activity by using CAG promotor-expression system. Fusion-responsible epitope on GPs was found to locate in the neutralization (FRNT)-relating epitope. Therefore, the recombinant GPs was considered to possess neutralization-and fusion-relating epitopes. In addition, hantavirus nucleocapsid (N) protein was also expressed in mammalian cells by using same vector. As the result the N protein may suppress that expression and transportation of the envelope protein. It was also shown that the virus like particle was not formed by GPs and N protein alone.2. By using recombinant GPs, pseudotype vesicular stomatitis virus (VSV) enveloped with hantavirus GPs altered to VSV G protein was produced (VSVΔG*HTN). Similar pseudotype VSV was produced with recombinant GPs of Seoul virus (VSVΔG*SEO). These pseudotypes were applied to rapid neutralization assay as safety alternatives of authentic viruses. These pseudotype virus particles were also applied to vaccination as alternatives to authentic virion. Mice were immunized with soluble recombinant GPs or pseudotype virion. In mice immunized with soluble recombinant GPs, FRNT antibody was not detected. Contrary, in mice immunized with VSVΔG*HTN virion, FRNT antibody was detected. Challenge administration of hantavirus was carried out by s.c. inoculation of 4 FFU of hantavirus. These mice did not show the elevation of anti-N antibody and hantavirus specific CD8 T cell response, indicating that the FRNT antibody could protect mice from hantavirus infection. On the other hand, all control mice immunized with VSVΔG*G or PBS could not escape from hantavirus infection. These results indicated that packaged recombinant GPs on VSV particle were possible to induce protective FRNT antibody. This study shows that novel approach for the development of vaccination of viruses that have difficulties in preparation of authentic virus particles.