Development of flavivirus vaccine including Japanese encephalitis virus by recombinant DNA technologies

构建了含有乙型脑炎病毒包膜蛋白preill、M、e蛋白编码序列的重组杆状病毒和痘苗病毒。这些重组蛋白在每个编码序列的上游都有一个信号序列，产生具有生物活性和抗原性活性的乙脑病毒蛋白，这些蛋白被糖基化并加工成与真实蛋白相似的蛋白。我们的研究结果还表明，重组病毒有效表达E蛋白需要编码在E蛋白上游的信号序列，当E蛋白的每个表位成功表达后，该蛋白在重组病毒感染细胞的表面和培养上清液中被检测到。重组病毒感染细胞释放的E蛋白为小颗粒、M蛋白低聚物或异聚物。感染这些重组体或接受这些重组体产物的动物对这些病毒表现出类似的免疫反应。更多的蛋白质，包括显著的中和抗体的产生，作为乙脑病毒感染和小鼠攻击测试成功地完成。通过对乙脑病毒单克隆逃逸突变体和株的分析，确定了E蛋白上的乙脑病毒特异性和WN亚群病毒特异性中和表位和株特异性表位的位置。与只含有特定表位的乙脑病毒E蛋白部分融合的hbsag成功地以颗粒形式产生。因此，我们可以开发出制备具有特异性表位的颗粒E蛋白和HBsAg颗粒的方法来有效地诱导中和抗体。这些结果为利用重组病毒技术开发乙脑及其他黄病毒的新疫苗提供了基础信息。少

Recombinant baculo- and vaccinia viruses containing the coding sequences of the viral envelope proteins, i. e., preill, M, and E protein of Japanese encephalitis virus (JEV) were constructed. These recombinants which had a signal sequence at the upstream of each coding sequence of the proteins produced biologically and antigenica-lly active JEV proteins which were glycosylated and processed as similar to the authentic proteins. Our results also indicate that efficient expression of the E protein by recombinant virus requires the signal sequence which is encoded upstream of E protein and when each epitope of E protein is successfully expressed, this protein is detected on surfaces and culture supernatant of recombinant virus infected cells. The E proteins released from recombinant virus infected cells were small particles and oligomers or heterooligomers with M protein.Animals infected with these recombinants or received the product of them showed similar immune response against these p … More roteins including remarkable production of neutralizing antibodies as JEV infection and challenge testing was successfully done by using mice. The E protein oligomer and hetero oligomer particles composed with M protein were more good immunogens and elicited biologically active antibodies such as neutraling and hemaggulutination inhibition than monomer E.Locations of JEV specific and WN subgroup virus specific neutralizing epitopes and strain specific epitopes on the E protein were detenaned by analyzing monoclonal escaped mutants and strains of JEV. HBsAgs fused with a part of JEV E protein containing only specific epitopes were successfully produced in particulate forms.As a conclusion, we could develop the methods which could produce particulate E proteins and also HBsAg particles with only specific epitopes to induce neutralizing antibody effectively. From these results we could show the basic informations to develop new vaccines for JEV and other flaviviruses by recombinant virus technologies. Less