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

1.利用CAG启动子表达系统表达的重组汉坦病毒囊膜糖蛋白G1和G2具有细胞融合活性。GPs上的融合负责表位位于中和（FRNT）相关表位。因此，重组GP被认为具有中和和融合相关的表位。此外，汉坦病毒核衣壳（N）蛋白也在哺乳动物细胞中表达。因此，N蛋白可以抑制包膜蛋白的表达和转运。病毒样颗粒不是由GPs和N蛋白单独形成的.利用重组GPs，制备了假型水泡性口炎病毒（VSVΔG*HTN）。用首尔病毒的重组GP（VSVΔG*SEO）产生类似的假型VSV。将这些假型作为真实病毒的安全替代品应用于快速中和试验。这些假型病毒颗粒也作为真实病毒体的替代物应用于疫苗接种。用可溶性重组GP或假型病毒体免疫小鼠。用可溶性重组GPs免疫小鼠，未检测到FRNT抗体。而VSVΔG*HTN病毒颗粒免疫小鼠，可检测到FRNT抗体。汉坦病毒的激发给药通过s.c.接种4FFU汉坦病毒。这些小鼠没有显示抗N抗体和汉坦病毒特异性CD8 T细胞应答的升高，表明FRNT抗体可以保护小鼠免受汉坦病毒感染。而VSVΔG*G或PBS免疫的对照小鼠均不能逃避汉坦病毒感染。这些结果表明，包装在VSV颗粒上的重组GPs有可能诱导保护性FRNT抗体。这项研究表明，新的方法，用于开发疫苗接种的病毒，有困难的制备真正的病毒颗粒。

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.