Chimeric Lyssavirus Glycoproteins with Increased Immunological Potential

The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH(2) half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against parental viruses in mice, whereas, pG-PVIII (site III part only) and pGPV-Mok did not. The site III part of PV (GT1) was a strong inducer of T helper cells and was very effective at presenting the site II part of various GTs. Both parts are required for correct folding and transport of chimeric G proteins which have a strong potential value for immunological studies and development of multivalent vaccines. Chimeric plasmid pGEBL1-PV broadens the spectrum of protection against European lyssavirus genotypes (GT1, GT5, and GT6).

狂犬病病毒糖蛋白（G）分子可被柔性铰链（flexible hinge）划分为两个部分：NH₂端半段（位点II部分），包含抗原位点II（antigenic site II），直至涵盖表位VI[epitope VI，aa264]的线性区域（linear region）（氨基酸[amino acids，aa]253至275）；以及COOH端半段（位点III部分），包含抗原位点III（antigenic site III）与跨膜与胞质结构域（transmembrane and cytoplasmic domains）。本研究通过细胞转染（cell transfection）与小鼠DNA免疫（mouse DNA-based immunization）实验，利用由某一基因型（genotype，GT）的位点II部分与同一或另一GT的位点III部分融合得到的同源及嵌合G基因（chimeric G genes），探究了两部分各自的结构与免疫学功能。我们测试了PV（巴斯德病毒株，Pasteur virus strain）狂犬病病毒（GT1）、Mokola（GT3）以及EBL1（欧洲蝙蝠狂犬病病毒1型[European bat lyssavirus 1，GT5]）的G基因之间的多种位点II-位点III组合。结果显示，质粒pGPV-PV、pGMok-Mok、pGMok-PV与pGEBL1-PV可在神经母细胞瘤细胞（neuroblastoma cells）中瞬时表达正确转运并折叠的抗原，且能在小鼠体内诱导出针对亲本病毒的病毒中和抗体（virus-neutralizing antibodies）；而pG-PVIII（仅含位点III部分）与pGPV-Mok则未观察到上述效果。PV（GT1）的位点III部分是T辅助细胞（T helper cells）的强诱导因子，且可高效呈递不同GT的位点II部分。嵌合G蛋白的正确折叠与转运需要两部分协同参与，这类蛋白在免疫学研究与多价疫苗（multivalent vaccines）开发中具备极高的应用潜力。嵌合质粒pGEBL1-PV可拓宽针对欧洲狂犬病病毒基因型（GT1、GT5及GT6）的保护谱。