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

1.利用CAG启动子-表达系统证实了重组汉坦病毒囊膜糖蛋白G1和G2(GPs)具有细胞融合活性。融合相关表位位于中和相关表位(FRNT)。因此，重组的GP被认为具有与中和和融合相关的表位。此外，汉坦病毒核衣壳蛋白(N)在哺乳动物细胞中也得到了表达。因此，N蛋白可能会抑制包膜蛋白的表达和运输。研究还表明，病毒样颗粒不是由GPs和N蛋白单独形成的。用重组糖蛋白制备了汉坦病毒G蛋白修饰的伪型水疱性口炎病毒(VSVΔG*hTN)。用汉城病毒的重组GP(VSVΔG*SEO)产生与之相似的伪型VSV。将这些伪型作为正品病毒的安全替代品应用于快速中和试验。这些伪型病毒颗粒也被用于疫苗接种，作为正宗病毒粒子的替代品。用可溶性重组糖蛋白或伪型病毒粒子免疫小鼠。可溶性重组GPs免疫小鼠后，未检测到FRNT抗体。反之，ΔG*HTN病毒粒子免疫的小鼠，可检测到FRNT抗体。汉坦病毒的挑战管理由S.C.接种4FFU的汉坦病毒。这些小鼠未出现抗N抗体升高和汉坦病毒特异性CD8T细胞应答，表明FRNT抗体对汉坦病毒感染具有保护作用。而ΔG*G和PBS免疫的对照组小鼠均不能逃脱汉坦病毒的感染。这些结果表明，包装在VSV颗粒上的重组GP有可能诱导保护性FRNT抗体。这项研究为发展疫苗接种难以制备正宗病毒颗粒的病毒提供了新的途径。

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.