NEW APPROACHES TO VACCINES AGAINST THE TICK-BORNE ENCEPHALITIS VIRUS COMPLEX

以前，构建了活的嵌合黄病毒，其含有 蜱传脑炎病毒（TBEV）结构蛋白CME或ME基因 其余的基因来源于登革4型病毒（DEN 4）。 的 ME嵌合体保留了其TBEV亲本小鼠的神经毒力， 它的M和E基因是来自，但它缺乏外围的， TBEV的侵袭性。 ME嵌合体进行突变， 分析，试图减少或消除神经毒力表现， 病毒被直接接种到大脑中。 三种不同的突变 独立地与小鼠 神经毒性 这些突变消除了：（i）TBEV PreM切割， 位点，其是M蛋白的适当加工所需的;（ii）TBEV E（包膜糖蛋白）糖基化位点;或（iii）第一DEN 4 NS 1 （非结构蛋白1）糖基化位点。 三个 减毒突变体在猴和蚊子中的生长都受到限制 细胞 重要的是，这些减毒突变体的肠胃外接种 诱导小鼠对致死性脑炎的完全抗性， 随后用高神经毒性ME嵌合体进行攻击。 这些 观察结果表明，一种新的策略，发展减毒活TBEV 疫苗 与高毒性TBEV不同，野生型Langat病毒（TP 21 株），所有TBEV复合体黄病毒中毒性最低的，具有低的 致脑炎潜力和外周毒力， 与任何人类疾病有关 以试图 确定TBEV复合病毒减毒的分子基础， 野生型病毒（TP 21株）基因组序列等 测定了其衍生的LGT减毒株（ES株）。 在蜱传黄病毒中，LGT基因组的长度不同， 与TBEV或Powassan病毒（一种TBE病毒）相比， 北美）基因组。 TP 21和E5基因组序列分析 揭示了多聚蛋白中的六个氨基酸差异，其中之一是 E蛋白第387位的Asn > Asp取代，这可能是 负责E5病毒的减毒。减弱突变， 通过LGT菌株的遗传分析鉴定的菌株将被引入 嵌合LGT/DEN 4和TBEV/DEN 4基因组和子代病毒将被 分析免疫原性和毒力丧失。

Previously, viable chimeric flaviviruses were constructed that contained tick-borne encephalitis virus (TBEV) structural protein CME or ME genes with the remaining genes derived from dengue type 4 virus (DEN4). The ME chimera retained the neurovirulence for mice of its TBEV parent from which its M and E genes were derived, but it lacked the peripheral invasiveness of TBEV. The ME chimera was subjected to mutational analysis in an attempt to reduce or ablate neurovirulence manifest when virus was inoculated directly into the brain. Three distinct mutations were independently associated with marked reduction of mouse neurovirulence. These mutations ablated: (i) the TBEV PreM cleavage site which is required for proper processing of M protein; (ii) the TBEV E (envelope glycoprotein) glycosylation site; or (iii) the first DEN4 NS1 (non-structural protein one) glycosylation site. Each of the three attenuated mutants was restricted in growth in both simian and mosquito cells. Significantly, parenteral inoculation of these attenuated mutants induced complete resistance in mice to fatal encephalitis caused by subsequent challenge with the highly neurovirulent ME chimera. These observations suggest a new strategy for developing a live attenuated TBEV vaccine. Unlike the highly virulent TBEV, the wild type Langat virus (TP21 strain), the least virulent of all TBEV-complex flaviviruses, has low encephalitogenic potential and peripheral virulence and has not been reported to be associated with any human disease. In an attempt to identify the molecular basis for attenuation of TBEV-complex viruses, the sequence of the genome of wild type virus (TP21 strain) and a more attenuated strain of LGT derived from it (strain ES) were determined. Among the tick-borne flaviviruses, the LGT genome differs in length of its 3' noncoding region compared to TBEV or Powassan virus (a TBE virus of North America) genome. Analysis of the TP21 and E5 genome sequences revealed six amino acid differences in the polyprotein, one of them is a substitution Asn > Asp in position 387 of E protein, which probably is responsible for attenuation of E5 virus. Attenuating mutations which were identified by genetic analysis of the LGT strains will be introduced into chimeric LGT/DEN4 and TBEV/DEN4 genomes and progeny viruses will be analyzed for immunogenicity and loss of virulence.