Experimental evaluation of Japanese encephalitis vaccine candidates for human use generated by recombinant technology

基于高度减毒牛痘病毒（NYVAC-JEV）和金丝雀痘病毒（ALVAC-JEV）的重组乙型脑炎（JE）候选疫苗在小鼠体内诱导特异性抗体和细胞毒性T淋巴细胞（ctl）的能力进行了评估。6至8周龄雄性Balb/c小鼠，每只小鼠以1 * 10^7 PFU的剂量接受1或2次腹腔接种这些乙脑候选疫苗，产生中和抗体和包膜(E)和非结构1 （NS1）蛋白抗体，通过放射免疫沉淀测定。这两种候选疫苗均可诱导乙脑病毒特异性T淋巴细胞在感染性病毒和/或非感染性病毒抗原刺激下增殖。小鼠中和抗体和乙脑病毒特异性记忆T细胞在NYVAC-JEV免疫后至少6个月和ALVAC-JEV免疫后至少4个月保持可检测水平。活病毒刺激后诱导增殖的细胞含有特异性CD8^+ ctl，这些ctl可裂解感染乙脑病毒的Balb/c小鼠原代肾细胞和感染表达膜前（prM）、E和NS1蛋白的重组痘苗病毒感染的P815肥大细胞瘤细胞。这些ctl也能裂解表达prM和E的痘苗重组病毒感染的P815细胞，以及表达E和NS1的痘苗重组病毒感染的P815细胞，但不能裂解仅表达NS1的重组病毒感染的P815细胞，说明ctl主要识别E，而不识别NS1。结果表明，重组乙脑疫苗NYVAC-JEV和ALVAC-JEV均能诱导小鼠产生乙脑病毒特异性抗体和ctl。

Recombinant Japanese encephalitis (JE) vaccine candidates based on a highly attenuated vaccinia virus (NYVAC-JEV) and a canarypox virus (ALVAC-JEV) were evaluated for their ability to induce specific antibodies and cytotoxic T lymphocytes (CTLs) in mice. Six- to eight-week-old male Balb/c mice that received one or two intraperitoneal inoculations with these JE vaccine candidates at a dose of 1 * 10^7 PFU per mouse, produced neutralizing antibody and antibodies to the envelope (E) and non-structural 1 (NS1) proteins as determined by radioimmunoprecipitation. Immunization with either of these vaccine candidates also induced JE virus-specific T lymphocytes that proliferated in response to stimulation with infectious virus and/or non-infectious viral antigens. Mice maintained detedtable levels of neutralizing antibody and JE virus-specific memory T cells for at least 6 months after immunization with NYVAC-JEV and for 4 months after immunization with ALVAC-JEV.Cells induced to proliferate after stimulation with live virus contained specific CD8^+ CTLs that lysed primary Balb/c mouse kidney cells infected with JE virus and P815 mastocytoma cells infected with a recombinant vaccinia virus expressing the premembrane (prM), E,and NS1 proteins. These CTLs also lysed P815 cells infected with vaccinia recombinants expressing prM and E,and those expressing E and NS1, but did not lyse P815 cells infected with a recombinant virus expressing only NS1, indicating that the CTLs mainly recognized E,but did not recognize NS1. These results demonstrate that both recombinant JE vaccines, NYVAC-JEV and ALVAC-JEV,induce JE virus-specific antibody and CTLs in mice.